Annu. Rev. Microbiol. 1990.

44:69-88
Copyright 1990 by Annual Reviews Inc. All rights reserved
PN'NK\ LLPIhL
IKL1\PZP1IP, L7KLIP,
PPL KLLP1IP
Thomas J. Chambers, Chang S. Hahn, Ricardo Galler,
l
and
Charles M. Rice
Depatment of Molecula Microbiology, Washington University School of Medicine,
Box 8230, 660 South Euclid Avenue, St. Louis, Missouri 63110-1093
KEY WORDS: positive-strand RNA virus. protein processing. animal virus. glycoprotein.
protease
CONTENTS
OVERVIEW............................................................................................ 650
INTRODUCTION TO FA VIVIRUS BIOLOGy.............................................. 650
Classiication and Life Cycle in Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
Importance as Disease Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
Replication in Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 652
ORGANIZATION AND TRANSLATION OF THE GENOME RNA ..................... 653
General Features ............................................. .................................... 653
Translation Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
PROTEOLYTIC PROCESSING OF THE VIRAL POLYPROTEIN........................ 655
The StruclUra[ Proteins . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 658
The Nonstructural Proteins . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . .. 658
PROPERTIES OF VIRAL PROTEINS........................................................... 660
Structural Proteins. . . .... . . . . . . ..... . .. . . . . . . . . ... . . . . . . . ... ... . . . . . . . . ...... . . . . . . . . . . . ..... . . . . . 660
Nonstructural Proteins........................................................................... 667
VIRION STRUCTURE AND ASSEMBLy...................................................... 671
CONSERVED RNA SEQUENCES AND STRUCTURES.................................... 673
5' and 3' Terminal Secondary Structures ................................................... 674
Conserved and Repeated Sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 675
RNA REPLICATION ....................e .................. ......................................... 675
Ipennanent address: Fundacao Oswaldo Crz, Departmento de Bioquimica e Biologia
Molecular, Rio de Janeiro, R CEP 21040, Brazil
69
0066-4227/90/1001-0649$02.00
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Quick links to online content
Further
ANNUAL
REVIEWS
650 CHAMBERS ET AL
COMPARATIVE ASPECTS ... ..+....... .................. ....... ......... ......................... 677
Genetic Divergence among Flaviviruses . + + - ..... ............... .... + ........ ....... ......... 678
Similarities H Other Positive-Strand Viruses...... ....... . ......... ................ ......... 679
CONCLUDING REMARKS AND FUTURE PROSPECTS.................................. 679
OVERVIEW
Flaviviruses are small, enveloped animal viruses containing a single positive
strand genomic RNA. Modem studies on these viruses began with the discov
ery, nearly a century ago, that the disease yellow fever was caused by a
filterable agent and transmitted to humans by mosquitoes (see 109). This
review summarizes recent developments in flavivirus molecular biology,
emphasizing the viral genome structure and the expression and possible
functions of the viral proteins. Highlights of the last several years include: (a)
a complete map of the flavivirus-encoded proteins and an emerging picture of
the processing events involved in their production; (b) extensive sequence
comparisons allowing the identification of potentially important structural
motifs in the genomic RNAs and encoded proteins; (c) the derivation of a
structural model for the major virion envelope protein; (d the recovery of
infectious favivirus RNA from molecularly cloned eDNA. In citing the
literature, preference has been given to recently published material. The
reader is referred to two books (143, 144) for comprehensive reviews of
earlier work.
INTRODUCTION TO FLA VIVIRUS BIOLOGY
Classification and Life Cycle in Nature
The Flaviviridae (from the Latin favus. or "yellow," referring to the pro
totype virus, yellow fever virus) was recently established as a separate family
(178), distinct from the Togaviridae, and currently includes 68 members (12).
The majority are arthropod-bore, transmitted to vertebrates by chronically
infected mosquito or tick vectors. However, isolates from bats and rodents
without known insect vectors have also been identified. The life cycle of the
arthropod-bore flaviviruses involves complex relationships among insect
vectors, vertebrate reservoirs, humans, and the environment (reviewed in 19).
Flaviviruses demonstrate antigenic cross-reactivity that i s strongest i n the
hemagglutination inhibition assay (HI); the highest specificity is shown in
neutralization (NT) assays ( 1 28). Cross-neutralization using polyclonal anti
sera is the basis for the separation of faviviruses into eight antigenic com
plexes; members of any serocomplex commonly share a range of biological
properties ( 1 2) (Figure 1). The four mosquito-bore complexes include the
Japanese encephalitis (JE; for favivirus abbreviations see Figure 1) group,
Ntaya, Uganda S, and the DEN groups. The tick-bore complexes include the
TBE and the Tyuleniy groups. Complexes without identified arthropod vec-
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FAVIVIRUS GENOME STRUCTURE & EXPRESSION 651
Antgenic cmplex 1ypmember or membrs with Arthropod Available suene O
(numbr 0Imembrs) 8vl80Icseuene vector
Potcin NucIccAdd
N-terminal C-terminal
Tick-eiti,,(12) Obmecitis bW) T 7 9.10
FmEstmeht|im(IEFE) T 127,127a,188
RoBmvo(6) RoBnvo
Jap eepalilis (10) Jap eophitis l} M 107.IS7 6.107.IIS.120.IS7.160
8jm18U) M 28.147,149 148 2
MJYrJl
M W 1258.9.9
5tDouephts (SLE) M 4.131 10,166
WestNile(WN) M 15.16,121,172 121 10,11,15.16,17.171.172,173
Tleiy (3) Tyulliy T
Ntaya(5) Ntaya
M
UpmsS(4) UjzdzS M
DS"(4)
DgIgI(D8N:) M 23,105
Dge typ 2 (DEN2)
M 4.S.'I.I84 184 6.37.38.51.58.60.77 .187
Dptp1(D6N1)
M 124
iS"t, 4(DEN4) M 97.190
M(5) M
U
UN 17 Yellow fever (YF) M 4.20.21.131 33,3,50.57.58.132.133
Figure 1 Flavivirus antigenic complexes, arthropod vectors, and sequence data. Antigenic
groups are talken from Reference 12. Arthropod vectors: T, tick; M, mosquito; U, unknown;
asterisk, arthropod vectors for some members of these groups have not been identified. The
ungrouped fla.viviruses include mosquito- and tick-transmitted viruses as well as some with no
known vector. Included are literature citations of published flavivirus protein or nucleic acid
sequence data.
tors include the bat-associated Rio Bravo group and the rodent-associated
Modoc group. In addition, 17 viruses, including YF, demonstrate neutraliza
tion propelties that do not allow their classification within these serocom
plexes. Relationships among faviviruses, based upon comparison of nucleic
acid and protein sequence data, closely follow those deduced from cross
neutralization tests using polyclonal antisera (see below). However, rela
tionships that do not fit the traditional classification can be defned at the
epitope level using monoclonal antibodies and alterative serologic tech
niques (68).
Importance as Disease Agents
Although humans are primarily dead-end hosts for maintenance of flavivir
uses in their natural cycle (182), arthropod-bore flaviviruses are responsible
for significant human and animal disease worldwide (110, l1 2a, 182). Clinic
al disease is expressed as fever, encephalitis, or hemorrhagic fever and
includes se:veral entities of major global concer, including yellow fever
(11 1 ), dengue fever with its associated dengue hemorrhagic fever (DHF) and
shock syndrome (DSS) (reviewed in 61,62), and Japanese encephalitis (112).
TBE, Kyasanur Forest disease, WN, SLE, and MVE are other important
agents of regional endemic or epidemic disease (110) . Disturbances in the
vector-vertebrate equilibrium have resulted in significant interegional spread
of these viruses. Worldwide eradication of favivirus pathogens is unlikely
because thly are maintained in animal reservoirs and can be transovarially
transmitted in arthropods. Thus far, vaccination is only available for YF,
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652 CHAMBERS ET AL
using the attenuated 17D strain (162), and for TBE and JE using inactivated
virus (reviewed in 68) .
Replication in Cultured Cells
Cells derived from many mammalian, avian, and arthropod sources can
support flavivirus replication in cell culture (80). The early events in flavivir
us infection are not well characterized and cellular receptors for faviviruses
have not been identified. Evidence suggests that the virus enters cells by
receptor-mediated endocytosis (43), presumably mediated by the virion en
velope protein (E) and plasma membrane receptors. In addition, in the
presence of subneutralizing concentrations of antibody, Fc receptors also
mediate attachment and uptake (43). The latter entry mechanism, termed
antibody-dependent enhancement, may play a role in development of DHFI
DSS in sequential infections with different DEN serotypes (reviewed in 61,
62) . Ultrastructural studies of WN infection of P388Dl cells, and of YF and
KUN infection of Vero cells, have localized single virions and virion aggre
gates to clathrin-coated pits on the cell surface; uptake into coated vesicles
rapidly follows attachment (45 , 78, 1 19). Virions are later found in uncoated
prelysosomal vesicles, where an acid-catalyzed membrane fusion is thought
to release the nucleocapsid into the cytoplasm (45). Consistent with this
release, a conformational change in the viral E protein (see below), possibly
important for fusion, occurs at low pH (55, 81) . Acid pH can also promote
fusion of WN virions with liposomal membranes or at the plasma membrane
(4), although this mode of entry does not lead to productive infection (45,
81). Ultrastructural analysis of the entry of mosquito-passaged JE and DEN2
viruses into mosquito cells or human monocytes suggests that direct plasma
membrane penetration can occur (63), although whether this initiates a pro
ductive infection is not clear.
The uncoating of virions, translation of the infecting virion RNA, and the
initiation of RNA replication have not been studied. Viral RNA synthesis can
be detected at three to six hours with the release of infectious virus beginning
at approximately 12 hours. Although growth kinetics appear similar in ver
tebrate and arthropod cells, maximal titers in different cell types can vary
considerably (114) . Even during the peak of virus production, a major
inhibition of host macromolecular synthesis is not observed (9, 176, 177). In
vertebrate cells, dramatic cytopathic and ultrastructural changes can be
observed, including vacuolation and proliferation of intracellular membranes
(11 3); infection is commonly cytocidal, although some vertebrate cell types
do not show these effects and become chronically infected. Arthropod cells
may demonstrate cytopathic effects, induding syncytium formation (re
viewed in 9, 167); generally infections are noncytopathic (114), and persistent
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 653
infections are more easily established in arthropod cells than in vertebrate
cells.
ORGANIZATION AND TRANSLATION OF THE
GENOME RNA
General Features
The genome RNA of faviviruses is single-standed and approximately 11
kilobases in length (reviewed in 9, 134, 176, 177). The genomic RNA is
infectious, and thus of positive polarity encoding the viral proteins necessary
for RNA replication. Genome-length RNAs appear to be the only virus
specifc mRNA molecules in favivirus-infected cells. The genomic RNA has
a type I cap at its 5' end (m7GpppAmp) and lacks cap-associated and interal
base-methylated adenine residues. The 5' cap is followed by the conserved
dinucleotide sequence AG. Genomic RNAs of mosquito-bore faviviruses
appear to lack a 3'-terminal po1y(A) tract (9, 134, 176, 177) and instead
terminate with the conserved dinucleotide CU. TBE RNA probably ter
minates with the dinucleotide AC (100, 127a), but the presence of additional
sequences or a poly(A) tract have not been rigorously excluded (C. Mandl &
F. X. Heinz, personal communication).
The most notable feature of the favivirus genome is the presence of an
open reading frame (ORF) of over 10,000 bases. ORFs for diferent favivir
uses (starting at the frst Met residue) range from 10,158 bases (DEN4) to
10,302 bases (MVE) and encode polyproteins of 3,386 to 3,434 amino acids
(compiled in Figure 2). No other conserved ORFs have been identifed in
either the genomic sense RNA or its complement. Flanking the long ORF are
5' (from 95 to 132 bases in length) and 3' noncoding regions (Figure 2). The
3' noncoding region of TBE (114 bases) appears to be dramatically shorter
than that of any of the mosquito-bore viruses (from 385 to 585 bases in
length) and lacks a number of RNA sequences and structures common to the
mosquito-bore viruses (see below).
Another interesting feature of favivirus genome RNAs is their high purine
content and low CG and UA doublet fequencies, the significance of which is
unclear. As discussed in detail elsewhere (133, 134), these nonrandom di
nucleotide frequencies and the resulting biased codon usage in the ORF may
infuence translation of the flavivirus genome and refect the adaptation of
these viruses to growth in their host environments.
Trnslation Strategy
The accumulated protein and nucleic acid sequence data for many faviviruses
(compiled in Figure 1 and discussed below) have shaped our current view of
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654 CHAMBERS ET AL
Favivirs (stain) 5' NC
OLN1 >67
DEN2{lAM) 96
OLN2|Ko1) -96
DENJ ?94
OLN4 101
M(OA582) 95
M{bcJu!) 95
KUN >75
N -
96
5LL
~b
TE(W) 132
Y 96
Y{1!D) 118
Y{A80) 118
OR
10,173
10,173
10,158
10,296
10,287
10,299
10,302
10,242
10,290
10,233
10 233
3' NC
gru0mc
50mc
lcny
105
454 10,723 37,38
443 10,712 60
124
385 10,644 97,190
585 10,976 157
583 10,965 66
>290 >10,664 Zb
>528 >10,926 32,90
166
114 10,488 99,100
574 10,960 15,16,17,
171,172,173
511 10,862 133
511 10 862 57
Figure 2 F1avivirus genome sizes. Listed are the lengths of the 5' noncoding regions (5' NC),
the long open reading frames (ORF), and the 3' noncoding regions (3' NC; includes the stop
codon terminating the ORF) for several flaviviruses.
viral protein synthesis. The order of proteins encoded in the long ORF is
5' -C-prM(M)-E-NS I -NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' . The structu
ral proteins C (capsid; the C precursor is called anchored C), M (membrane;
the M precursor is called prM) , and E (envelope) are encoded in the 5' quarter
of the genome and the genes for the non structural proteins are located in the
remainder. The non structural proteins include the large, highly conserved
proteins NS 1 , NS3, and NS5, and the four small hydrophobic proteins NS2A,
NS2B, NS4A, and NS4B. For a description of the protein nomenclature used
here and its relationship to previously used designations, see the following
references ( 1 33, 1 34, 1 77) .
N-terminal sequence data for the TEE, YF, WN, and SLE C proteins show
that translation can begin with the first AUG in the long ORF. Only 4 of the
1 1 faviviruses for which data are available have a purine in the -3 position
relative to this AUG, which is preferred for eukaryotic translation initiation
[CC(NG)CCAUGG] (85, 86) . The mosquito-bore viruses have a second
in-frame AUG between 1 2 and 1 4 codons downstream from the first AUG
with an A residue at the 3 position. For WN, N-terminal sequence analysis
of virion-associated C protein (see below) ( 1 6) suggests that this AUG may
also serve as a translation initiation site. In TBE, an additional short ORF of
24 codons is present 5 ' to the long ORF (C. Mandl & F. X. Heinz, personal
communication; 1 27, 1 88). Translation termination of the long ORF has no
strong termination codon preference; all three are represented: UAA (DEN4,
KUN, MYE, WN, TEE) , UAG (DEN2) , and UGA (YF).
Several experimental approaches led to the suggestion that some flavivirus
proteins might be produced from the viral mRNA by interal initiation of
tanslation. Alterative interretations of these experiments have been dis
cussed elsewhere ( 1 34, 1 77) . The curent view, given the presence of a single
ORF encoding at least 1 0 favivirus-specific proteins, is that translation
initiates near the 5' end of the genome and that individual viral proteins are
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FLA VIVIRUS GENOME STRUCTURE & EXPRESSION 655
produced by proteolysis. This view is supported by the available protein
sequence data (Figures 1 and 3), cell-free translation of genomic RNAs (see
below), the identification of high molecular weight polyproteins in infected
cells (21 , 26), and the recent translation mapping experiments using KUN
infected Yero cells, which demonstrated that the viral proteins are synthesized
in the order predicted by the genome sequence ( 145).
PROTEOL YTIC PROCESSING OF THE VIRAL
POL YPROTEIN
Based on N- and C-terminal protein sequence data and from the alignment of
homologous sequences (Figure 3), proteolytic cleavages separating the viral
polypeptides can be classified into distinct groups. The N termini of prM, E,
and NS 1 follow predicted signalase cleavage sites ( 168) contributed by the
C-terminal hydrophobic regions of anchored C, prM, and E, respectively. A
signal sequence also precedes the N terminus of NS4B, suggesting that this
hydrophobic protein is processed in association with endoplasmic reticulum
(ER) membranes. The N terminus of NS2A follows a cleavage site defined by
the sequence (Val-X-Ala; X=Ser, Thr, GIn, Asn, Asp) that fulfills the
( -1 , 3) mle for a signalase site; however, the site lacks a requisite upstream
hydrophobic region ( 1 68) . A protease of novel specificity has been invoked
for this ckavage (20, 147), although cleavage by signalase has not been
excluded. Additional cleavage within the NS2A region has been inferred (21,
102, 104; see below), but the cleavage sites have not been characterized.
Cleavage sites generating the N termini of NS2B, NS3, NS4A, and NS5 are
highly conserved among flavivimses (Figure 3). They occur after two basic
amino acid residues (Lys-Arg, Arg-Arg, or Arg-Lys) or occasionally afer
Gln-Arg (DEN2 and DEN4 NS3) and are usually flanked by short side-chain
amino acids, most commonly Gly, Ser, or Ala (Figure 3). A flavivirus
encoded protease was hypothesized to mediate such cleavages ( 1 33), a strat
egy common to several other RNA vimses (reviewed in 87) , and recent data
substantiating this belief are presented below.
Although rigorous proof is not available for many of the molecular events
in favivirus polyprotein processing, it seems likely that translation in associa
tion with the rough ER (RER), translocation, cleavage, acquisition of the
corect protein topology, assembly of the replication complexes, and virion
morphogenesis are all closely interconnected phenomena. The following
model (Figure 4) attempts to put currently available data into a single
framework. Beginning with translation of the C protein, signalase cleavages
mediate the primary processing events involving stmctural protein precursors.
The nascenlt C protein contains a C-terminal hydrophobic domain that acts as
an interal signal sequence for translocation of prM into the ER lumen, where
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656 CHAMBERS ET AL
Si-lik cleavages (RE lumn)
FY AC-pM pM-E E-NS! NS4A-NS4B
DEI LrTBmvFHLTTR VTr5N+HR0VU1 UWVQA4D5U0V1
DE 1PT+FHLTTR 1+ + VATH+NENGFL
DE LFATLA+FHLT5R VTP5HT+NRCVGV GV QA+DNGCV1
Om 1FT+F5L5TR VAP5YG+NRCVGV GFTVQA+DNGCV 1GL1A+NNGL1
M 1AYA+NK5NF AY54@ ATNVHA+DTGCA1 VGVA+NYGNL
KU 1AUVUA+VTL5Nt X@ + X+B
M 1GFAA+LKL5TF VAPAY5+FNCLGN ATNVHA4DTGCA1 VGLVA!NEYGM
SL 1GLA55+@ 1APAY5+@ AT5VQA+
W =J: @+@ +DTGAI VGAVAA!NEMGW
3 LLHTGG+VTLWK VGFA1S+[ 5LGVGA+@ VSV+@
1b V1TLLG+NTLAAT LAPVYA+5RCTHL TLGVGA+DVGCAV AGLVAJNEMGFL
Cleavages afer dibasic residues (cyoplasmc)
VVm Ah0LVmoL NSlA-NS2B NS2B-NS3 NS3-NSA NS4B-NS5
DEI H8K~~KR+5VNLL
m LNKKK+TAGN11 KT5K+5HLN VKKQK+ tAAG8F+SLTLNL TTSTKK+@
1NmK+T5L0LN
Om LNGR~--~KR+5T1TLL KGASRR.SWPLNE QVKTQR+5GALHD FASGR.SITLDI AQTPRGTGTTG
M KU8KQNKR+GGNG5 NFNKKR+GHPATE LKTTKK+GGWHD FAGKR+5A15F1 KFSLKR4GRPGGR
KU @+GGKTG1 + + + Z4
M MGKKQKKK+GG5T5 NNKm+GHFT LK1TKK+@ tGKR+SA1Gtt KFtKK+
5lL INRP--SKKR.GGTRSL HPNGKJSWPASE GKHSKRJG
W ]+GGTAGF DFN8KR+GHPATE LQYTKR+GGVLHD FA5GKR+5Q1GLV KPGLKR+GGAKGR
Y L55RKR+5HDVLT R1FGR+ VRGARR+ FGRR+@ HTGRR+
TE LQKKG~KR+5ATDHN AHRGR+SF5LFL LRS5RR+SDLVFS YASGRRJ5FGDVL ASGGRJGGSEGD
Oer cleavages
Me-NtoC pM-M NSI-NS2A
vvms mMH dibic, delaye, novel, sligtly delayed,
ptdase. cytosl Iw lwm
DEI N+NNQR 88RDK+5VL EENLVK5H5A+G5GEVD
DE2 N+NDQRK 8RRK+5VL
+
OE N+NNQR RRRDK+5VA
b N+NQ8FF KKR+5VLTF NHVK5QVTA+GQT5
M N+
1
5KR5RR+ TTLWQVDA+FNGENV
KU N+

SK5K+ EQ1l
m M4So SmS4SITvQT BSTLVkSkQA4FNGoMI
S I
58R5RR+515VQH EAV5RVTA+GVAGGN
W I
5R5RK+@ KTLVQ5RVNA+YNADN1
Y N+
5K8h+@ 5HLVKHVTA+
TE N+X

GSRTRRJSVLIPS QGLVSMV A!ONGELL

Figure 3 Flavivirs cleavage sites_ Sequences surrounding polyprotein cleavage sites (arrows)
for several flaviviruses are aligned_ Alignments based on N-terminal and C-terminal protein
sequence data (underlining) from a number of different flaviviruses (Figure 1)_ Strain-spcific
diferences surounding the cleavage sites were noted but not shown here. The most stiking
stain-specifc difference was the sequence RGG at the -4 to -2 positions preceding the NSI-2A
cleavage site in the Beijing-I strain of IE (66)_ In compiling these data, preference was given to
the first full-length published sequence for each serotyp_ References are as follows: DENI (105);
DEN2 (60); DEN3 (124); DEN4 (97, 190); JE (157); KUN (28); MVE (32, 90); SLE (166); W
(15-17, 171-173); YF (133); TE (99, 10)_
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 657
Processing of the tavivirus
polyprotein
UKl
J`NC
' _.

capsTRucURA NONSTRUCTURAL

-
--
+* + + ** . . . u
--
` ''

anchC prM E
u .'
Vlnon
I assembly?
C
virion
release?
NSI-2A NS2B
** V

NSI NS2A
NS3 NS4A-4B NS5
+?

NS4A NS4B
signalas, rapid, lumen
. dibasic, rapid, cytoplasm
. dibasic, delayed?, cytoplasm
U dibasic, delayed, lumen
V novel, slightly delayed, lumen
Figure 4 Schematic of the processing events for the tavivirus polyprotein. The top depicts the
viral genome; the structural and nonstuctural protein coding regions, the 5' cap, putative 3'
secondary structure, and the 5' ad 3' untranslated regions are indicated. Boxes below the
genome indicate precursors and mature proteins generated by the proteolytic processing cascade:
(shaded boxes) structural prteins, (open boxes) nonstructural proteins. Black bars represent
contiguous stretches of uncharged amino acids. A,terisks indicate probable N-linked glycosyla
tion sites for YF. See the text for a discussion of the data supporting this model.
the C-prM cleavage and core glycosylation of prM occur. Secondary cleavage
events of anchored C protein and prM may be linked to virion assembly and
release and are discussed in more detail below. At the C terminus of prM,
adjacent hydrophobic stetches interrupted by a charged residue act as a
stop-transfer sequence for prM and as a signal sequence for translocation of
the E protein. Two adjacent hydrophobic sequences at the C terminus of the E
act similarly for E protein stop-tansfer and NS 1 translocation. Translocation
of NS 1 into the lumen of the ER is followed by cleavage of E-NS 1 and core
glycosylation of NS 1 . The NS2A-2B cleavage is mediated in the cytosol by a
viral protease. Cleavage of NSI-2A appears to be delayed for some flavivir
uses and is hypothesized to occur in the lumen of a vesicular compartment.
Smaller NSI-2A-related forms have been detected, suggesting possible
alterative cleavages within the NS2A region prior to the cleavage generating
the C terminus of NS 1 . Processing of the NS2B-3-4-5 region is proposed to be
mediated primarily by a viral protease; the NS2A-2B, NS2B-3, NS3-4A, and
NS4B-5 cleNges occur in the cytosol; and signalase generates the NS4B N
terinus. This cleavage strategy is consistent with the cytoplasmic location of
NS3 and NS5 which are presumed enzymatic components of the viral
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658 CHAMBERS ET AL
replicase, and it suggests that the hydrophobic NS2A, NS2B, NS4A, and
NS4B proteins may be membrane-associated, perhaps via membrane
spanning domains. The experimental data supporting this model are summa
rized below.
The Structural Proteins
Analysis of the processing of the strctural region encoding C, prM, and E in
vivo and in vitro indicates a requirement for membranes and intact signal
sequences. In vitro translation of TBE virion RNA has shown that production
of viral C and E proteins in reticulocyte lysates requires a particulate fraction
from Krebs ascites cells, and that the activity is destroyed by detergent
treatment (158, 159) . These observations have been confrmed for DEN4
(101) , WN (121) , and YF (137) by translation of transcripts containing the
structural gene sequences in reticulocyte lysates with canine pancreatic micro
somes. C-terminal analysis of the WN C-related protein produced in the
presence of membranes (called anchored C) , reveals an 18-amino acid
hydrophobic segment at the C terminus not present in virion C protein (121) .
The corresponding region in DEN4 is necessary for correct expression of prM
in vitro; deletion of 10 amino acids from this sequence or interruption by
insertion of a nonconservative amino acid prevents glycosylation, transloca
tion, and correct cleavage of prM (101). Similarly, deletion of the
homologous YF sequence alters the prM-related in vitro products (137).
Cleavage of the WN prM and E-related proteins during cell-free translation in
the presence of membranes occurs at the same signalase sites used in infected
cells (121) . Furthermore, the hydrophobic sequence preceding the YF E
protein is necessary for tanslocation of this protein in vitro (137) .
Results consistent with signalase-mediated processing of the structural
protein region have also been obtained in flavivirus-infected cells and in
transient expression systems. Using vaccinia virus recombinants, DEN4
structural proteins can be expressed in the absence of downstream nonstructu
ral sequences (8, 41) . Recombinants lacking the signal sequence preceding
the E protein do not express this protein, although substitution with a
heterologous signal sequence allows expression (8) . Furthermore, inhibition
of KUN structural region processing in cell culture is observed in the presence
of the leucine analog threo-t-hydroxyleucine, which is known to inhibit
signalase cleavage when incorporated into signal peptides ( 31).
The Nonstructural Proteins
PROCESSING OF THE NSI-2A REGION Analyses of NSI processing in flavi
virus-infected cells and in transient expression systems indicate that a signal
sequence preceding the N terminus is required and that NS2A may play
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FLA VIVIRUS GENOME STRUCTURE & EXPRESSION 659
a role in NS 1 processing. In cells infected with DEN4-vaccinia recombinants,
the signal sequence, but not the putative stop-transfer sequence in the C
terminus of the E protein, is required for NSI expression (8, 41) . In YF
infected cells, evidence for an NS 1-2A precursor to NS 1 (21) suggests that the
NS2A-2B cleavage or alterative cleavages in the NS2A region occur prior to
the NSl -2A cleavage. NSl-2A has not been identified for DEN2 (181) or
TBE (91) , but in IE-infected cells, an additional 56-kilqdalton (kd) glycosy
lated NS I -lA-related protein (NS 1' ) is present and secreted with similar
kinetics to those of the 48-kd NSI protein (102) . A role for NS2A in the
NSl -2A cleavage is also suggested by studies with DEN4-vaccinia recom
binants in which progressive C-terminal deletions of the NS2A region gener
ate uncleaved but glycosylated NSI -2A-related proteins (41) . NSI glycosyla
tion may also be important for NSI -2A cleavage because tunicamycin treat
ment alters the production and secretion of JE NS I -related proteins (102) and
generates aberrant cleavage products of the YF NSI -2A polyproteins (21).
PROCESSING OF THE NS2B-3-4-5 REGION Comparative sequence data ( 3,
46) (see section on properties of viral proteins below) and recent in vitro and
in vivo studies implicate the N-terminal third of NS 3 as a serine protease
responsible for mediating some of the nonstructural-region cleavages that
occur after dibasic residues (see section on NS3 properties). Translation of
RNA transcripts in reticulocyte lysates showed that polyproteins containing
the proposed NS 3 protease domain are capable of dilution-insensitive cleav
age at the authentic NS2A-2B and NS2B-3 cleavage sites, and that amino acid
substitutions of the proposed active-site residues in the NS3 protease domain
often abolish processing (T. J. Chambers, R. C. Weir, A. Grakoui, D. W.
McCourt, J. F. Bazan, R. J. Fletterick, & C. M. Rice, submited) . Cleavage
at the N termini of NS4A and NS5, which occurs afer similar double basic
cleavage sites, has not yet been convincingly demonstrated in vitro.
Data from studies of viral proteins synthesized in infected cells are also
consistent with this processing scheme and implicate a serine protease for
some of the nonstructural protein cleavages. DEN2-specific proteins of appar
ent molecular weight (MW) 100-220 kd, which accumulate in the presence of
N-4-tosyl-phenylalanyl-chloromethylketone and ZnCh, appear to be pre
cursors of smaller proteins (26). The arginine analog canavanine alters the
expression of DEN2 NS3, and proteins of MW 115 and 130 kd are detected
(125). Several YF-specific polyproteins can be detected using region-specific
polyclonal antisera (21). These have the predicted MWs for NS3-4-5 (210
kd), NS4AB-5 (140 kd), NS4B-5 (130 kd), NS3-4A (110 kd), and NS4AB
( 39 kd). These data suggest that site-specific cleavages occur on the non
structural polyprotein at the proposed double basic sites.
The N-terminal amino acid sequences for KUN and YF NS4B suggest that
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660 CHAMBERS ET AL
signalase may catalyze this cleavage (20, 147) , although membrane involve
ment has not been investigated experimentally. This intriguing observation
suggests that the cleavage event generating the N terminus of NS4B occurs in
the lumen of the RER and that NS4A and NS4B contain membrane-spanning
domains.
In summary, processing of the flavivirus polyprotein involves a remarkable
progression of cleavages mediated by proteases of both host and viral origin.
After translation of the anchored C hydrophobic sequence, the flavivirus
polysome presumably becomes associated with the RER, and translation of all
viral proteins is likely to be membrane-associated. Further experiments to
clarify many of the details of processing and to elucidate the actual membrane
topologies of favivirus proteins are greatly needed.
PROPERTIES OF VIRAL PROTEINS
Processing of the favivirus polyprotein generates at least 1 0 membrane
associated viral proteins (three structural , seven non structural proteins) , and
two polypeptide cleavage fragments. The following section summarizes cur
rent information regarding the structure, maturation, and possible functions of
these polypeptides (Figure 5) .
Structural Proteins
THE CAPSID PROTEIN The C protein present in virions is a small (predicted
MW 1 2-1 4 kd), highly positively charged protein, (27% Lys+Arg for YF)
( 1 34), which forms a structural component of the nucleocapsid. Flavivirus C
protein sequence homology is low, but regions of hydrophobic and hydrophil
ic amino acids are conserved, including a C-terminal hydrophobic domain
that is immediately preceded by hydrophilic region, and a central
hydrophobic region (99). The N-terminal region contains hydrophilic sec
tion that in mosquito-bore faviviruses is interrupted by a hydrophobic insert
not present in TBE.
The favivirus C protein exists i n a number of different forms due to both
N- and C-terminal modifcations. N-terminal sequence data (Figure 3) indi
cate that the initiating methionine is removed, presumably by cellular ami
nopeptidase. Other possible N-acetylated N termini would not have been
detected in these studies. In fact, C proteins retaining the initiating methionine
have been detected for WN ( 1 6) and SLE (4) , and some WN C proteins also
begin at the second in-frame methionine at position 1 6 of the ORF ( 1 6, 1 21 ) .
For virion C proteins of WN and KUN, the C terminus has been positioned
afer a double basic cleavage site 1 8 amino acid residues from the prM N
terminus (Figure 3) ( 1 21 , 148) .
Differences in the electrophoretic migration of intracellular C proteins,
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range of
N- c-
%
aNiVi
preicted hydrophobic
poi
PI
trinal terminl
rsidues
MWs
cleavage clava
(YF)
aC 12.3-13.7 M
I
s 4
!
virio C 10.5-11.7 M I 37
prM 18.1-19.0 S
I
S 39
pr 9.8-10.7 S
I
DL 31
M 8.2-8.5 DL I S 48

E 53.3-5.3 S S 41
NSI 39.2-.0 S ?L 3
NS 23.7-25.4 ?L I 56
NS 13.8-14.5 I I 5
NS3 68.5-6.5 D I I 39
NSA 16.016.4 D S 56
NSB 26.5-2.9 S
I
I 49
NS5 103-10 D
i
NA 39
I
. ! -. -
nt membra-
charge asiat
(YF) d?
+25
y
+26 n
+5 y
+1 n
+4
y
-5
y
-7 n?
+ ?
-7 ?
0 n
- 2 ?
+3 ?
+14 n
N-1i1
gly?
additio propries
no
!
hmoN W,mK
hyiN

hC:Hw,bo,
no
I Wnl_id p
I 0mm
y
I M ==
y
I
MOOm
mN-mes mM
n
i WNN
! =mMQW
!
h Om,W
I
IIZ=de1
ye C I mj Vee
I
p
y
mn
NId; CF ae
n
b bc poOm
n
bbc poI
n
! h 1
n
I
b b poOm
n
l
bbc poOm
I
I
h wLm
n
.
psible ftction
KAb; n1""id
@pM .igs
ug7. ely mo7
vmwmgei
oa
?
vqs c bg M
at
mu: r b;
m f mj vlI
pe e
wm7;pc
ae
NS1_?
mln
a
gh
mM
.

.
WKApy?
Jl_r 5 Pperies of flavivirus proteins. The ranges of polypptide MWs for each of the flavivirs plypptides, as prdicte from available sequence data 8 given.
Lalization and proteases (or specifcity) respnsible for cleavages generating the N ad C termini (see text and Figures 3 and 4) 8 abbrviated as follows: cellular methionine
amino pptidase (M); host signalase (S); afer dibasic residues, cytoplasmic (C) or lumen (DL); novel spcifCity, lumen (?L). T prnIhydrophobic amino acid content (Ala,
Ph. lie. Leu. Met. Va. Trp. Tyr) and net charges of tese plypptides are given for YF. See text for fher details.
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662 CHAMBERS ET AL
virion-associated C proteins, and C proteins produced in cell-free systems
have been observed. Intracellular KUN C protein migrates faster and lacks a
tryptic peptide compared with virion capsid (176) . In contrast, for WN and
TBE, intacellular C protein migrates more slowly than the virion-associated
form and lacks a tryptic peptide (159, 170) . Recently the difference between
intracellular and virion-associated WN C protein was not detected, but the
form produced in vitro in the presence of membranes, which contains the
C-terminaI hydrophobic domain, migrated slower than the virion-associated
form lacking this domain (121). Similar results were observed for the TBE C
protein produced by cell-free translation, although sequence data was not
obtained (158) . The C protein has been difcult to detect in DEN4 and YF
cell-free translation studies (101, 137). The intracellular YF C protein de
tected after short pulse-labelling also migrates slower than the virion
associated form, although the structural difference has not been characterized
(21). A TBE variant (Tyrolian stain ZZ/9) contains a virion-associated C
protein that migrates faster than those of other TBE strains, possibly because
of a nonconservative amino acid substitution (56, 99). The precise roles of N
and C-terminal modifications in these electrophoretic differences and in the
function of the C protein remain unclear.
THE prM PROTEIN The prM protein is a glycoprotein precursor (predicted
MW 18. 1-19.0 kd, modified by carbohydrate addition) to the structural
protein M. This precursor undergoes a delayed cleavage to form M and the
N-terminal pr segment, the fate of which is unknown. This cleavage is
presumably linked to virus maturation or release, as prM and M are found on
intracellular and extracellular virions, respectively (see below).
N-terminal amino acid sequence data (Figure 3) indicate that the N ter
minus of prM immediately follows a signalase site (10 1, 121) contributed by
the C terminus of the capsid protein. The N terminus of the M protein
immediately follows a pair of basic amino acids believed to represent a
cleavage site for either a viral or host protease. Available C-terminal sequence
data suggest no further truncation of this protein (Figure 3).
The N-terminal pr segment is predominantly hydrophilic, containing six
conserved cysteine residues all of which participate in disulfde bridging
(122), and a variable number of potential N-linked glycosylation sites (Figure
6). The C-terminal M segment, present in mature virions, contains a short
ened ectodomain (41 amino acids) and retains the two potential membrane
spanning domains.
The location of potential N-linked glycosylation sites (Asn-X-Ser/Thr) for
prM is shown in Figure 6. Serologically related viruses show position con
servation of these sites: Asn69 for the DEN serogroup or Asn15 for the JE
serogroup. YF contains three potential sites within the pr region, and TBE a
single site. All of these sites display the sequence Asn-X-Thr. Additional
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 663
prl1I E NS!
--
:
.
DEN!
-. ! ! ! !
DEN2 -. ! 2 ! 2
DEN3 -. ! !
(
!
)
DEN4 -. ! i
(
!
)
! !
J
1- ! ! 2
KU
1 !H
MVE
1 ! !U
SLE
L. ! : !U
W
1- !U
YF
l. ' (3 !
3
TE
.. ! (:) !
S
Figure 6 Potential N-linked glycosylation sites. The positions (drawn to scale) of potential
N-linked glycosylation sites in prM, E, and NSI are indicated (diamond-headed lollies). Potential
N-linked sites enclosed in parentheses are probably not utilized because of their location in
hydrophobic and membrane-associated domains. Open symbols indicate potential sites of the
sequence AsnPro-Thr, Asn-Pro-Ser, or Asn-Asp-Thr that are often poor acceptor sites for
N-linked glycans (84). A dotted line indicates the cleavage site in prM used to produce M, and
thick lines indicate the portions of prM and E believed to be membrane-associated. Data were
taken from the references in Figure I.
Asn-X -Thr sites are found in the hydrophobic C-terminal domain of M in YF
and SLE.
Data suggest one N-linked glycan i n prM for DEN2, DEN4, KUN, and
WN, and two in YF; the attachment sites are not known. Studies of endogly
cosidase H sensitivity show that DEN2 and KUN prM contain approximately
two kd of mannose-rich glycan, and glycopeptide analysis reveals structural
heterogeneity (146, 185, 186). DEN4 prM in infected cells or expressed by
vaccinia recombinants appears to contain a single mannose-rich glycan (8,
191), although it contains two such glycans when produced in a cell-free
system, presumably at Asn69-Leu-Thr and Asn32-Lys-Cys ( 1 01). The biolo
gical relevance of glycosylation at the Asn-X-Cys site remains in question
given the in vivo results and the fact that all six cysteine residues participate in
intramolecular disulfide bridges (122). YF prM contains two N-linked gly
cans (21). WN prM produced in cells and cell-free systems contains mannose
rich glycans ( 1 21 ) , although the number of chains has not been determined.
Glycosylation of prM presumably has an important role in virion assembly
and release (discussed below).
THE E PROTEIN The E protein (predicted MW 53-54 kd) is the major
structural protein of the virion-glycosylated forms are found in some but not
all faviviruses. This protein presumably plays a role in a number of biological
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664 CHAMBERS ET AL
P

Figure 7 E protein model. Circles indicate amino acids in the TBE E sequence; absolutely
conserved positions or those with only conservative substitutions among mosquito-bore virses
are filled or shaded. respectively. Disulfide bridges are shown. and diamonds indicate potential
N"linked glycosylation sites (see Figure 6). Residues changed in TBE or YF monoclonal antibody
neutalization escape mutants are indicated by arrows (and the selecting monoclonal antibody)
and arrowheads. respectively. Thin lines denote hypervariable regions and the thick line a highly
consered domain in E proposed to be involved in fusion. See text for further details. This fgure
is adapted from the TBE E protein model published by Heinz and coworkers (70, 98) with
additional information derived from other flaviviruses as cited in the text and Figure 1.
activities including virion assembly, receptor binding, and membrane fusion,
and is the major target for neutralizing antibodies (reviewed in 68) .
The N and C termini of E are produced by signalase cleavages; no evidence
indicates additional trimming of the polypeptide backbone (Figure 3). Its
primary structure, mode of synthesis, and studies of protease digestion of
virions indicate that E is a typical membrane protein with a large N-terminal
ectodomain anchored in the bilayer via a hydrophobic C-terminal domain.
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FLA VIVIRUS GENOME STRUCTURE & EXPRESSION 665
Comparison of deduced E protein sequences reveals areas of striking homolo
gy as well as divergence (70, 99, 134) (Figure 7). The 1 2 Cys residues in the
E ectodomain are strictly conserved and, in the case of WN, are all involved
in intramolecular disulfide bonds (122) (see below) .
A major recent advance is the derivtion of two-dimensional E protein
structural models. These models assume a generally conserved structure for
favivirus E proteins and are based on (0) determination of the intramolecular
disulfide bridges for WN ( 1 22), (b) examination of topological and biological
properties of E epitopes defined by monoclonal antibodies (reviewed in 68;
see also 13, 18, 39, 49, 67, 71, 82), (c) physical and immunological
characterization of E protein fragments derived by protease or chemical
cleavage (55, 98), (d localization of antibody binding sites to deleted forms
of the JE E protein expressed in bacteria (103, 104), (e) nucleotide sequencing
of neutralization-escape mutants of TBE and YF (92, 98) , and if) reactivity of
anti-peptide sera with native E from MVE ( 1 36) .
Three domains, stabilized by disulfide bridges, were originally defined
from WN E protein disulfide bonding data (122). These include an N-terminal
region containing five strongly hydrophilic segments, a central region, and a
C-terminal region. These three regions are linked by less-conserved disulfide
free regions, and a hydrophobic anchor is present at the C terminus. Assum
ing that this patter of disulfide bridge formation is a general feature of
flavivirus E proteins, the model has served as a framework for incorporating
immunological and structural data obtained from a number of diferent favi
viruses.
The most extensive studies have utilized the TBE E protein and the
nomenclature proposed for TBE will be used here (see Figure 7). Heinz and
coworkers (:reviewed in 68, 70) characterized three major epitope clusters (A,
B, and C domains) in the E ectodomain by utilizing a panel of monoclonal
antibodies in competitive-binding assays. All three domains contain epitopes
involved in neutralization and hemagglutination inhibition, but only domain
A contains flavivirus cross-reactive epitopes. The experimental approaches
outlined above have allowed these domains to be correlated with particular
subregions of the E protein. Domain A, which is derived from a disulfde-rich
region of residues 50-125 and the region of residues 200-250 (Figure 7)
contains variable regions as well as highly conserved sequences (residues
98-111) possibly involved in acid-catalyzed fusion (70, 136) . Several neutra
lization-escape mutants map to this domain for both TEE (98) and YF (92) .
TBE domain A epitopes are sensitive to denaturation by SDS, reducing
agents, or trypsin, as might be expected from the secondary structure model
(55) . This domain also undergoes a pH-dependent conformational change
(55) consistent with its possible role in endocytotic fusion events.
Domain 13 consists of a trypsin-resistant fragment comprising residues
300395 and is enriched for TBE complex-specific epitopes. These epitopes
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666 CHAMBERS ET AL
are sensitive to reducing agents, but not SDS, acid pH, or trypsin-treatment
(55). Using E subregions from JE expressed in bacteria, the region between
residues 303-396 has also been shown to contain both JE-specific and sero
group cross-reactive neutralizing epitopes dependent upon a disulfde cross
linkage between residues 304 and 335 (103, 104). Two studies have provided
evidence for the biological importance of this domain in modulating
pathogenicity in vivo. A neutralization-escape mutant of TBE that maps to
Tyr384 (B4, Figure 7) is attenuated in mice and confers protection against
lethal challenge (cited in 70) . Similarly, a change at ASP390 selected durng
passaging of MVE in human SW -13 cells also appears to lead to attenuation in
mice (95). These data suggest that this region may be involved in tissue
topism and perhaps effect E-host receptor interactions.
TBE domain C is composed of a disulfide-free loop, and epitopes in this
domain are not denatured by SDS-treatment but are protease-sensitive (55).
The TBE glycosylation site, conserved among faviviruses, maps to position
154 (closed triangle in Figure 7) within this domain (70). Elimination of this
glycan renders some domain C epitopes sensitive to SDS denaturation (55).
Experimental results utilizing antipeptide sera for the MVE E protein are in
general agreement with the TBE model and contribute additional insights
( 1 36). Peptides generating antisera with highest neutralizing activity map to
the N-terminal region, suggesting that this region may be exposed. Further
more, competition experiments with monoclonal antibodies and antisera
raised to E peptides from MVE subregions corresponding to the A and B
domains of TBE predict that these domains interact.
The role of N-linked glycosylation in E function is unclear. Potential
N-linked glycosylation sites are not strictly conserved, but, in number and
position, similarities are noted among serogroup members (Figure 6) . A site
near position 1 50 is conserved among all DEN serotypes, some members of
the JE subgroup and TBE, but not YF. Indirect evidence exists for the
utilization of this site in TBE (70, 1 79) . An additional site at Asn67 is found
for the DEN group. YF contains a potential site at Asn309 also found in SLE.
Sites located near the C terminus (Figure 6) are probably not good acceptors
because of their localization in the E protein hydrophobic domain (84).
Neither of the sequenced strains of WN and KUN viruses have predicted
N-linked glycosylation sites of the Asn-X-SerlThr type (28, 1 72) . It is
therefore surprising that labelled mannose can be incorporated into cell
associated E but not the KUN extracellular virion E ( 1 83). This finding may
refect strain differences, but could also refect utilization of potential AsnlOr
X-Cys acceptor sites and consequent disruption of disulfde bond formation.
Several studies examined flavivirus E glycosylation in both vertebrate and
mosquito cell cultures (39, 91 , 146, 179). Evidence exists for both simple and
complex glycans (39, 91 , 146, 179, 1 91 ) with apparent host-specifc dif-
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 667
ferences (TBE) (152, 179) as well as diferences depending upon the stage of
E maturation (102) . Diferences in E protein glycosylation have also been
noted for South American and African strains of YF (39) . Glycosylation and
release of the JE E protein has been examined in detail ( 1 02) . Newly
synthesized, cell-associated E, produced in either monkey kidney or mosquito
(C6/ 36) ceills, contains a single mannose-rich endo H-sensitive glycan. Vi
rion-associated E is slowly released into the culture medium (tll2
> 6-8
hours) and the endo H-resistance of extracellular virion-associated E, from
both cell types, indicates that glycans have been converted to complex forms
prior to VifUS release. E release is impaired but not abolished in the presence
of tunicamycin, indicating that E glycosylation may be important for some
faviviruses . For TBE virions, however, carbohydrate removal does, not
reduce infectivity for avian cells (179), although glycosylation may still play
some role in virion assembly or release.
Nonstructural Proteins
THE NSI PROTEIN The NSI glycoprotein exists as cell-associated (reviewed
in 177), cell-surface ( 1 41 , 177), and extracellular nonvirion (91 , 1 02, 146,
181) forms. Flavivirus infections elicit antibodies to NS1 with complement
fixing (CF) activity and the secreted form of this protein has been called the
soluble complement fixing (SCF) antigen ( 138, 146) . Type-specific@ com
plex-specific and group-reactive epitopes (reviewed in 71) have been defined
for NSI and appear to play a role in protective immunity. Active immuniza
tion with NSI or passive immunization with anti-NSI monoclonal antibodies
protects animals against challenge with homologous faviviruses (8, 14, 48,
140--142, 189) .
NSI has a predicted MW of 39-41 kd, with additional modification by
N-linked glycosylation. Amino acid sequence data indicate that the N ter
minus is produced by signalase cleavage (Figure 4) and the C terminus by
cleavage at a novel protease site located upstream of the cleavage site
originally proposed for YF NS 1 ( 1 33). The protein includes 1 2 strictly
conserved cysteine residues (except DEN4) (97), invariant glycosylation
sites, and additional regions of high sequence homology. A Cys-Trp motif,
commonly found at the active site of thiol proteases (161), is located near the
NSI C terminus, although no evidence exists for an NSI -associated protease
activity.
Multiple forms of NS 1 have been detected and differences exist between
faviviruses. For IE and YF, a protein containing NSI and a portion of NS2A
can be found within cells (21, 102, 104) and extracellular fuid (102) . A
putative NSI dimer has been detected ( 1 81 ) within cells and in extracellular
fuid of DEN, SLE, and Powassan. This 86-kd complex can be dissociated to
produce monomeric NS 1 by boiling or treatment with acid pH but not by SDS
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668 CHAMBERS ET AL
(without boiling), urea, or reducing agents. Possible NSI dimers have also
been detected for JE and YF, but they appear to be disrupted under more mild
conditions (P. Mason, personal communication; I . Muylaert, R. Galler, & c.
Rice, unpublished data) . I n pulse-chase experiments, the DEN2 NSI dimer
appears after 200 minutes, suggesting that posttranslational processing may
be important for dimer formation ( 1 80) .
The physical differences between the membrane-associated and secreted
forms of NS I are not clear. Membrane association of NS 1 has been inferred
from the ability of NS I -specific monoclonal antibodies to lyse virus-infected
cells in the presence of complement ( 1 41 ) . In addition, two secreted fors
have been reported; a slowly sedimenting form and a more rapidly sediment
ing form (91 , 102, 1 38, 1 81 ) . Treatment of JE NSI from the culture medium
with nonionic detergent slows its sedimentation rate, suggesting possible
membrane association of even secreted forms ( 1 02) . Experiments to examine
the hydrophobic properties of the protein have yielded conflicting results. For
DEN2, newly synthesized monomeric NSI behaves as a hydrophilic protein
becoming more hydrophobic upon dimerization ( 1 80). In contrast, cell
associated JE NS 1 and NS 1 -2A (NS 1 ' ) behave more like hydrophilic proteins
( 102) .
Carbohydrate modification of NS 1 is implied by the conserved patter of
N-linked glycosylation sites. All mosquito-bore flaviviruses contain two
conserved sites (Figure 6) , with an additional site in all JE-complex members
except JE. A different patter exists for TBE, which shares one of the
conserved sites, but has two additional unique sites. Direct evidence for
glycosylation of NS 1 comes from several studies. Cell-associated DEN2 NS 1
contains approximately four kd of N-linked oligosaccharide, which consist of
structurally heterogeneous mannose-rich glycan, probably distributed over
both conserved N-linked glycosylation sites ( 1 46) . Both endo H-sensitive and
resistant carbohydrate has been detected on DEN4 NS 1 expressed by vaccinia
recombinants ( 1 91 ) . For IE, the cell-associated and extracellular NSI and
NS l ' from mammalian cells and the cell-associated forms from insect cells
have two N-linked oligosaccharide chains ( 1 02) . For both cell types, the
oligosaccharides of cell-associated NS 1 are mannose-rich; extracellular forms
from mammalian cells contain at least one complex glycan, indicating that
terminal addition occurred before release. These glycoproteins are released
slowly (t
1 l2
> 6h) from mammalian cells but are not secreted from insect
cells; secretion is abolished by tunicamycin. DEN2 NS 1 from mammalian and
insect cells contains two N-linked oligosaccharide chains; in contrast to JE
NS 1 , cell-associated and extracellular forms contain both mannose-rich and
complex carbohydrate ( 102, 1 81 ) . For TBE virus, both the cell-associated
NSI and secreted NSI contain fucose and galactose, suggesting the presence
of complex type glycans (91 ) .
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 669
A stuctural model incorporating the current data for the NS 1 glycoprotein
is not available and the relationship of epitopes to the primary structure of
NSI is poorly understood. Antigenic domains have been defned at both the N
and C-termini of JE NSI ( 1 04) . Monoclonal antibody epitopes of DEN2 have
been localized to a region between amino acids 273-346 that contains a highly
conserved sequence (residues 325 to 337) in a cysteine-rich region ( 1 29) .
THE Ns2A PROTEIN The NS2A protein is the frst of four relatively small
hydrophobic proteins (NS2A, NS2B, NS4A, NS4B) found within the NS2
and NS4 subregions of the polyprotein. A series of hydrophobic regions are
conserved among faviviruses ( 1 34) in position but not sequence, suggesting
that these proteins are membrane-associated. NS2A has been implicated in the
processing of NSI (see above) . The apparent MW of NS2A after extraction
and analysis by SDS PAGE is lower than the predicted 24-25 kd. The
N-terminal sequences of the KUN, YF, and DEN2 NS2A proteins (Figure 3)
indicate that the N terminus of NS2A may result from cleavage by an enzyme
of novel spe:cificity. The known C-terminal sequence of KUN NS2A indicates
that the proltein is not truncated (Figure 3). Two forms of the YF NS2A (MW
20 and 22 kd) have been observed; the structural basis for this heterogeneity is
unknown (20).
THE Ns3 PROTEIN NS3, the second largest viral protein (predicted MW
68-70 kd), is highly conserved among flaviviruses ( 1 00, 1 34) and contains no
long hydrophobic stretches. A role for NS3 in viral RA replication has been
postulated, and recent sequence comparisons (see below) suggest that it is
probably at least bifunctional, containing both a protease activity and possibly
a nucleotide triphosphatase/helicase activity. These properties suggest a
cytoplasmic: localization for NS3, with membrane association via interactions
that have not been defned.
Comparison of the NS3 N-terminal region (residues 1-1 80) with cellular
serine proteases has revealed significant homology in four regions of NS3 (3,
46) (Figure 8) . Hiss3, ASP77, and Serl38 comprise the putative catalytic triad
of the YF NS3 protease active site. The conserved motif Gly-X-Ser-Gly-X
Pro, found at YF NS3 amino acids 1 361 41 and at homologous positions in
other faviviruses represents the sequence surrounding the serine protease
nucleophilic serine (46) . The conserved Asp\ 31 residue, followed by either
Tyr, Phe, or Leu for all flaviviruses, and the conserved sequence Gly-Leu
Tyr-Gly-Asn-Gly (NS3 residues 1 51-156 for YF) , is hypothesized to form
pat of the substate binding pocket that in part determines cleavage specific
ity. As disl;ussed earlier, the viral protease usually cleaves after Arg, but
unlike trypsin, prefers dibasic residues surrounded by amino acids with short
side chains. Protease domains are encoded by a number of positive-strand
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670 CHAMBERS ET AL
, , ,
DEN2 ( 4 3) EGTFHTMWBVTRG [ l3aa] WADVKKDLISYGGGW [ 43aa] SLDFSPGTSGSPIVD KKG KWGLYGNGV
DEN4 ( 43) EGVFHTMWHVTRG [ 13aa] WADVRNDMISYGGGW [ 43aa] TLDFKPGTSGSPI IN RKG KVIGLYGNGV
JE ( 43) ENVFHTLWHTTRG [ l3aa] WGSVKEDRIAYGGPW [ 43aa] SLDYPRGTSGSPILD SNG DIIGLYGNGV
KUN ( 4 3) EGVFHTLWHTTKG [ l3aa] WGSVKEDRLCYGGPW [ 4 3aa] TLDFPTGTSGSPIVD KNG DVIGLYGNGV
MVE ( 43) EGVFHTLWHTTRG [ 13aa] WGNVKEDRVTYGGPW [433) SLDYPIGTSGSPIVN SNG EIIGLYGNGV
WN ( 4 3) EGVFHTLWHTTKG [ 13aa] WGSVKEDRLCYGGPW [ 43aa] TLDYPTGTSGSPIVD KNG DVIGLYGNGV
YF ( 45) GGVFHTMWHVTRG [ 13aa] WASVKEDLVAYGGSW [ 4 4aa] ALDYPSGTSGSPIVN RNG EVIGLYGNGI
TBE ( 46) KGVLHTMWBVTRG [ Baa] WADVREDVVCYGGAW [ 4 3aa] PIDLVKGTSGSPILN AQG VVVGLYGNGL
BVDV ( 1740) QGGISSVDBVTAG [ 26aa] EYGVKTDSGCPDGAR [ 40aa] DLKNLKGWSGLPIFEASSG RVVGRVKVGK
HCV ( 1 650) QGG lS SVDBVTCG [ 26aa] EYGVKTDSGCPEGAR [ 40aa] DLKNLKGWSGLPIFEASSG RVVGRVKVGK
HeCV \ ? ! NGVCWTVYHGAGT [ l3| YTNVDNDLVGWPAPQ | 4 T| PISYLKGSSGGPLLCP AG HAVGIFRAAV
SAP ( 4 3) KDTLLTNKBVVDA [ 3l| KYSGEGDLAIVKFSP [b| DLSTTGGNSGSPVFNEKN EVIGIHWGGV
SGT ( 29) QDIVLTAAHCVSG [ 34aa] YNGTGKDWALIKLAQ [ 73aa] GVDTCQGDSGGPMFRKDNADEWIQVGIVSWGY
TRP l 32) SQWVVSAAHCYKS [ 33aa] SLTINNDIMLIKLKS [ 7 6aa] GKDSCMGDSGGPVVC SG KLQGIVSWGS
CHT ( 34) ENWVVTAAHCGVT [ 34aa] SLTINNDITLLKLST [ 76aa] GVSSCMGDSGGPLVCKKNG AWTLVGIVSWGS
ELA ( 37) QNWVMTAABCVDR [ 37aa] DVAAGYDIALLRLAQ [ 78aa] VRSGCQGDSGGPLHCLVNG QYAVHGVTSFVS
SIN ( 133) EGKVMKPLHVKGT [ lldd] TKSSAYDMEFAQLPV [ 35aa] RGVGGRGDSGRIMD NSG RVVAIVLGGA
SGPA ( 25) VAHALTAGBCTSN [ 12aa] TSFPNNDYGIIRHSN [ 65aa] NVCAQPGDSGGSLFA GS TALGLTSGGS
SGPB ( 25) TYYFLTAGBCTDG [ 19aa] SSFPNNDYGIVRYTN [ 61aa] NVCAEPGDSGGPLYS GT RAIGLTSGGS
ALP ( 28) TKGFVTAGBCGTV [ 16aa] RVFPGNDRAWVSLTS [ 63aa] NACMGRGDSGGSWITS AG QAQGVMSGGN
Chymo 8 57 102 195 210
YF NS3 # 53 ?? l3B l5l
Figure 8 The NS3 protease domain. Aligned sequences of flavivirus NS3 proteins and cellular
proteases surrounding the putative catalytic triad (arrows) of the serine protease domain. Also
shown are sequences from two pestiviruses (numbered from the beginning of the ORF) , bovine
viral diarrhea virus (BVDV) (30) and hog cholera virus (HCV) (l08), hepatitis C virus (HeCV)
(75), and the autoprotease domain in the C terminal region of the alphavirus capsid protein for
Sindbis (SIN) virus (59, 135). Numbers refer to the distance of the first residue shown from the N
termini of the proteins. Abbreviations for the cellular proteases are trypsin (TRP); chymotrypsin
(CHT); elastase (ELA); Streptomyces griseus trypsin (SOT); Streptomyces griseus proteases
(SOPA and SOPB); Streptomyces aureus protease (SOP); Lysobacter enzymogenes a-lytic
protease (ALP). See the text and References 2, 3, and 46 for furher details.
RNA viruses, including an autoprotease domain in the alphavirus capsid
protein, the pestivirus p1 25, the potyvirus nuclear inclusion protein, and the
picoravirus 3C and 2A proteases. The latter three are cysteine proteases,
with Cys replacing Ser as the active site nucleophile (see 2, 3, and 46 for
review and specific references) .
NS3 also contains signifcant regions of homology with the D-E-A-D
family of RA helicases (47). This patter is also present in the homologous
proteins of other positive-strand RNA viruses and in proteins with nucleotide
triphosphate-(NTP) binding motifs. The sequence alignment of these various
proteins generates seven regions of amino acid conservation, designated I,
lA, and II-VI (47). These segments are located between residues 1 91 -508 of
YF NS3, C-terminal to the NS3 protease domain. Based on the common
sequence motifs for binding sites A and B of known NTP-utilizing enzymes
and putative helicases, segments I and II are proposed to be these sites for the
NS3 protein. Segment I contains the conserved motif Gly-X-Gly-Lys-ThrlSer
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FLAVIVIRUS GENOME STRUCTURE & EXPRESSION 671
(residues 201-205 for YF) and segment II the motif Asp-Glu-Ala-His/Asp
(residues 289-292) . Another highly conserved region, corresponding to seg
ment VI (residues 444--508), may be a nucleic acid binding site (47). Seg
ments IA and III-V are less conserved regions of unknown function. No
evidence supports cleavage of the NS3 protein into separate protease and
helicase domains.
THE Ns2B, Ns4A, AND Ns4B PROTEINS These small proteins (Figure 5),
like NS2A, are poorly conserved among faviviruses ( 1 00, 1 34) , but contain
similar strultural features consisting predominantly of multiple hydrophobic.
potential membrane-spanning domains. The NS2B and NS4B proteins are
readily identifiable in infected cells, but NS4A has been definitively identified
only for KUN ( 149). No posttranslational modifications of these proteins are
known, despite the presence of a conserved, potential N-linked glycosylation
site in the C-terminal portion of NS4B (90). These proteins may form
membrane components of the viral replication complexes and could be in
volved in membrane localization of NS3 and NS5 via protein-protein in
teractions.
THE NS5 PROTEIN NS5 is the largest (predicted MW 1 03-1 04 kd) and most
highly conserved of the favivirus proteins ( 1 00) . C-terminal sequence data
for KUN indicate that the protein extends to the predicted end of the ORF
( 1 48). NS5 is a basic protein and does not contain any long hydrophobic
stretches. The possible role of NS5 as the viral RNA polymerase (see below)
and the generation of its N terminus by cleavage in the cytoplasmic compart
ment (presumably by NS3 or an alterative protease) suggest that NS5 is
located in the cytoplasm, although it is associated with membranes.
Regions of sequence conservation are distributed throughout NS5 . A highly
conserved domain in NS5 (YF residues 531 to 675), contains the sequence
motif Gly-Asp-Asp ( 1 33, 1 34), which is present in nonstructural proteins
from a number of positive-strand RNA viruses and is believed to play a role in
RNA-dependent RNA synthesis (76, 79, 1 34).
VIRION STRUCTURE AND ASSEMBLY
Flavivirus virions contain a nucleocapsid (25-30 nm diameter) surrounded by
a lipid bilayer derived from host membranes containing the envelope proteins
E and M (o prM). The diameter of the virion is approximately 40 nm with
surface projections of 5-10 nm ( 1 13). Although morphologically in
distinguishable, two virion forms have been characterized; intracellular vi
rions contain only prM, whereas extracellular virions, released into the
culture fluid, contain predominantly M (for review see 1 38) (Figure 9).
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672 CHAMBERS ET AL
Intracellular Virion
Extacellular Virion
Nucleoapsid
Figure 9 Structure of intracellular and extracellular favivirs virions.
Ultastuctural studies indicate that virion morphogenesis occurs in associa
tion with intacellular membranes (reviewed in 1 1 3). Electron microscopic
studies of favivirus-infected vertebrate (78, 89, 106, 1 23) and mosquito (35,
65, 83, 1 1 6, 1 50) cell cultures, as well as in vivo studies in mouse brain (6,
1 51 ) have consistently observed morhologically mature virions first within
the lumen of the ER. In many studies, virions appear to accumulate within
disorderly arrays of membrane-bound vesicles. Budding intermediates and
clearly distinguishable cytoplasmic nucleocapsids have not been often
observed, suggesting that the assembly process occurs rapidly. Dramatic
alteration of the intracellular membranes is a hallmark of favivirus infection,
and the tansport of nascent virions, from the ER to the cell surface where
exocytosis occurs is believed to involve vesicles that may arise from the ER
(35) or other components of the host secretory system (65, 1 50) . Alterative
ly, dissolution of the vesicles may accompany cytolysis during late stages of
infection. Budding of virions through the plasma membrane has been
observed only infrequently (65, 1 06, 1 23, 1 50) and does not appear to be a
major mechanism for virion release.
These observations, together with the stctural information available for
the virion proteins and their mode of synthesis ( 1 21 ) , suggest a model for
virion assembly and maturation. The cytoplasmic, highly basic domain of the
C protein presumably interacts with viral genomic RNA to form a nucleocap
sid precursor. The hydrophobic segment of the anchored C protein may serve
to localize assembly of nuc1eocapsids to membrane sites ( 1 21 ) , and also
function as an interal signal sequence for prM ( 1 01 ) . Translocation of the
prM and E proteins positions their ectodomains within the lumen of the ER
and their hydrophobic C termini in the lipid bilayer. This orientation of C,
prM, and E with respect to the ER membrane suggests that nuc1eocapsids
acquire an envelope by budding into the lumen. The molecular interactions
involved in this assembly process have not been studied, but E and prM, as
present in intracellular and extracellular virus, appear to be associated as
detergent-stable heterodimers ( 1 74). Both prM and E are predicted to have
only small cytoplasmic domains, but unlike simple transmembrane anchors,
their hydrophobic C termini are highly conserved and could be involved in
E-prM or envelope-nucleocapsid interactions. This model predicts that the
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 673
anchored C cleavage occurs on the cytoplasmic side of the ER. In view of the
conserved basic amino acid pair preceding the virion C-anchored C cleavage
site, cleavage may be mediated by a viral protease, perhaps NS3. No infora
tion elucidates the timing of anchored C cleavage, which may occur im
mediately after tanslation or later during formation of the nucleocapsid.
Modification of E (for some viruses) and prM glycans by trimming and
terminal addition implies movement of newly formed virions through a
pathway similar to that used for synthesis of host plasma membrane glycopro
teins (21 , 102, 1 21 ) . A role for NS 1 in virion assembly, transport, or release,
as opposed to RNA replication, has been suggested because of its glycosy
lated and secreted forms.
The delayed cleavage of prM (see 138) and the luminal localization of the
cleavage site suggest that the event may occur in a compartment of the
secretory pathway, mediated by a host protease or possibly a viral protease
( 1 34) . The cleavage site sequence, Arg-X-Arg/Lys-Arg (where X is variable),
is similar to that recognized in maturation cleavages of numerous viral
glycoproteins (reviewed in 1 54) and cellular pro-proteins. The inability to
detect intracellular M-containing virions suggests that this cleavage occurs
just prior to or during release of virions. The biological role for prM cleavage
is unclear, but like similar glycoprotein cleavages for other enveloped viruses
( 1 69) , it may trigger changes in the virion envelope that promote infectivity.
In this regard, one study ( 1 74) reported that the specific infectivity of WN
intracellular virus is 60-fold lower than extracellular virus [which has a
specific infectivity of 600 particles per plaque-forming unit (PF)] . A
reorganization of the WN virion envelope has been reported to occur with
prM cleavage because detergent-stable E-M heteIdimers are not observed,
but instead apparently trimeric complexes of the E protein are seen ( 1 74,
1 75). In contrast to WN, cross-linking of detergent-solubilized extracellular
TBE results in preferential formation of E protein dimers (69). Although these
results suggest basic diferences in envelope structure between mosquito
bore and tick-bore faviviruses, further structural studies are needed before
frm conclusions can be made.
CONSERVED RNA SEQUENCES AND STRUCTURES
Short conserved RNA sequences and complex secondary stuctures play
important cis-acting roles in translational regulation as well as in RA
replication alnd encapsidation of a number of plant and animal RNA viruses.
Comparisons of favivirus sequences have led to the identification of several
potentially important elements. Figure 10 shows a schematic representing
these conserved RNA sequences and stuctures that is discussed below. Their
functions in virus replication, if any, remain speCUlative and need to be
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674 CHAMBERS ET AL
~~ ~~~~
uural rtcns Nsttutral rtcns
J'
5'C5
OLN --
OLN4 --
M
-:
m --
N
-
Y
--
L
J' NC
\
C52 C5l


.- --I- .I---
Figure 10 Conserved and repeated RNA sequences, A schcmatic of the flavivirus genome is
shown with the 5' noncoding (5 ' NC), the open reading frame (ORF) encoding the structural
proteins and non structural proteins, and the 3 ' noncoding region (3' NC) indicated. Shown below
and drawn to scale are the 5' and 3' noncoding regions of several flaviviruses highlighting
conserved (5' CS, CS 1 CS2) and repeated (similarly flled boxes) sequences as described in the
text. AUG codons or termination codons flanking the ORFs are indicated by ticks above or below
the lines, respectively. Also indicated is the short ORF in the 5' noncoding region of TBE.
Sequences were obtaincd from the sources given in the legend of Figure 3.
addressed by experimentation. None of the RNA features shared by the
genomes of mosquito-bore faviviruses have been identified in TBE.
5' and 3 ' Terminal Secondar Structures
Secondary structures, conserved in conformation and stability but not in
primary sequence, can be predicted for the 3 ' -terminal 90 bases of the
mosquito-bore viruses ( 1 1 , 50, 58, 157, 1 60, 1 71 , 1 90) . The predicted
stability (G) of this hairpin loop structure varies in different flaviviruses
from -40 to -60 Kcal/mol. Several lines of evidence (for review see 1 34;
also 1 1 , 58, 60) support the existence of such secondary structures. Stem-loop
structures have also been demonstrated directly at the 3' termini of several
families of plant viruses (cited in 58, 153) . For some viruses, such structures
are substrates for amino acylation in vitro and in vivo an
d
have been im
plicated in initiation of minus-strand RNA synthesis. Similar studies have not
been reported for flaviviruses.
Although stable stem-loop structures were not initially predicted for the
WN 5 ' -terminal region ( 1 7), Brinton & Dispoto ( 10) identified potential
secondary structures (G values from - 17 to -28 Kcal/mol) near the 5 '
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FLAVIVIRUS GENOME STRUCTURE & EXPRESSION 675
termini of several favivirus genomic RNAs including DEN2, DEN4, MVE,
SLE, WN, and YF (with the corresponding structures possible in the 3 ' end of
minus-strand RNA).
Conserved and Repeated Sequences
Two short conserved RNA sequences (called CSI and CS2; Figure 1 0),
shared by all mosquito-bore faviviruses, are located 5' to the putative 3 '
terminal secondary structure (58). CSI is about 26 nucleotides in length and
located immediately adjacent to the terminal secondary structure. Pa of CS 1
is complementary to a conserved sequence near the 5' end of the genome in
the region encoding the capsid protein (5 ' CS). Base-pairing of these or other
terminal sequences could lead to cyclization of of the viral genome that may
be important for regulating genome translation, replication, or packaging ( l0,
58, 1 57) . While evidence supports cyclization of alphavirus and bunyavirus
RNAs (cited in 58), the importance of such structures in replication is still
unclear. Cyclization of flavivirus RNAs has not been examined. The most
highly conserved sequence (CS2) is approximately 24 nucleotides in length
and is located 1 2 to 22 bases 5 ' to CS 1 . CS2 is duplicated in members of the
JE and DEN subgroups (Figure 10) .
Besides these short sequences that are conserved among flavivirus sub
groups, subgroup-specific features are also observed. YF has a set of three
closely spaced repeats found between nucleotides 10, 374 and 10, 520 ( 1 33).
These repeats, each about 40 nucleotides i n length, contain four to six
diferences in pairwise comparisons. Members of the JE subgroup have a
different sequence, about 25 nucleotides in length, present in two copies in
the 3 ' noncoding region. In contrast to YF, these repeats are separated by 1 20
to 1 30 bases. In the 5 ' noncoding region, WN contains three short sequences,
six to seven bases in length, repeated once ( 17) . Members of the JE subgroup
also show significant nucleotide sequence homology in their 5 ' noncoding
regions ( l0, 17) . TBE contains a duplicated sequence of about 20 bases
toward the 3 ' end of the genomic RNA; one of the repeated sequences is in the
region encoding the C terminus of NS5 and the other is in the 3 ' noncoding
region ( 100). It will be of interest to compare the sequences of more divergent
tick-bore faviviruses to see if other conserved RNA features can be identi
fied.
RNA REPLICATION
After translation of the incoming genomic mRNA, RNA replication involves
synthesis of complementary minus strands that are then used as templates for
production of additional genome-length plus-stranded molecules. These plus
strands can then be used for translation of structural and nonstructural
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676 CHAMBERS ET AL
polypeptides, minus strand synthesis, or be encapsidated into virions (re
viewed in 9, 1 67, 1 76, 1 77) .
In infected vertebrate cells, actinomycin D-resistant synthesis of favivirus
specifc RNA is detectable within six hours, and a progressive increase in
plus-strand genome-length RNA is observed. Evidence suggests that plus
strand RNA molecules are synthesized from genome-length minus-strand
templates by a semiconservative mechanism involving replicative in
termediates (RIs) and replicative forms (RFs) (25, 27) . RFs are defined as
duplex RNA molecules; RIs contain, in addition, nascent single-stranded
RNA molecules. RFs and RIs can be detected both in infected cells (25, 27)
and in the products of in vitro RNA polymerase reactions (24, 52) .
The RNase sensitivity of uniformly labeled RIs allows a prediction of the
average number of nascent single strands per RI ( 1 ) , and the values reported
range from -1 (KUN; 25) to 5 (DEN2; 27) . From 1 0 to 1 5 minutes are
required for completion of flavivirus genome-length products, which is about
l O-fold slower than the rate observed for poliovirus (25 , 27) . Interestingly,
differences between poliovirus and flaviviruses, in terms of the number of
nascent RNA molecules and the rate of elongation, correlate with the length
of their latent periods (27). Some mechanism must exist to regulate the
synthesis of plus-strand RNA relative to minus-strand RNA because a ratio of
plus to minus strand RNA of at least 10 to 1 has been observed at the peak of
DEN2 RNA synthesis (27). In contrast to alphaviruses, which shut off the
synthesis of minus strand templates (reviewed in 1 55) , minus strand synthesis
in DEN2-infected KB cells continues throughout the replication cycle (27) .
Although favivirus-specific proteins have been shown to sediment with
membrane fractions of infected cells ( 1 76, 1 77) , immunofuorescence studies
indicate a diffuse cytoplasmic distribution for NS3 and NS5, the presumed
replicase components ( 1 17, 1 1 8) . A specifc association of NS3 with microtu
buIes has also been suggested ( 1 1 8) . As judged by the localization of double
stranded RNA ( 1 77) and from cell-fractionation studies (24, 25, 52, 1 77) ,
viral RNA synthesis appears to be localized principally to the membranes of
the perinuclear endoplasmic reticulum. Subcellular fractions containing these
membranes are enriched in virus-specific RNA-dependent RNA polymerase
activity and have been used to optimize conditions for polymerase activity
before (24, 25, 52, 1 77) or after (54) detergent treatment.
The conservation of NS3 and NS5 among faviviruses and their homology
with helicases and polymerases, respectively, suggest enzymatic roles in
flavivirus RNA replication. Although both proteins are present in subcellular
fractions containing polymerase activity, most of the WN NS5 can be re
moved without signifcant loss of activity (53). These investigators (53)
pointed out the possibility that only small quantities of replicase-associated
NS5 are necessary for the observed polymerase activity. For Sindbis virus and
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 677
tobacco mosaic virus, the domains of the nonstructural proteins containing the
conserved Gly-Asp-Asp polymerase motif ( 1 34) are produced in small
quantities by readthough of in-frame termination codons.
Defective-interfering (DI) particles are valuable tools for the study of RNA
virus replication and may play a role in viral pathogenesis in some systems
(see below). For some viruses, strongly interfering DI particles are easily
generated by high multiplicity passage ( 1 26) and contain tuncated and rear
ranged genomic RNAs. These RNAs contain cis-acting sequences necessary
for replication and packaging but usually do not encode viral proteins. They
therefore need a helper virus to supply these functions in trans. In favivi
ruses, DI particles have been observed in persistently infected vertebrate cell
cultures, but strongly interfering DIs are not readily obtained under these
conditions or during serial high multiplicity passaging (9). Several possibili
ties may explain this observation, including the hypothesis that, under the
conditions tested, most of the virus-specifed components of the replication
machinery are required in cis. However, complementation at low levels has
been demonstrated between temperature-sensitive mutants of JE (40) or SLE
(74) that have defects in RNA synthesis.
A system in which DIs appear to be readily generated has been studied in
some detail by Brinton and coworkers (see 9 for review). Studies that began
with the observation of heritable susceptibility to YF virus in mice ( 1 39) led to
the discovery that a single autosomal dominant allele can confer resistance to
favivirus infection (see 9 for review). Flaviviruses can replicate in resistant
mice possessing this allele, but the spread of WN infection is slower and
significantly lower peak viremias are found (10
3
- to 1 0
4
-fold) than in congenic
susceptible mice. WN replication has also been studied in primary fibroblasts
from these strains. In cells from resistant mice, viral RNA synthesis was
reduced, lower titers of infectious virions were released, and a high propor
tion of DIs was found even after a single growth cycle. These results indicate
that a specifc, but as yet unidentified, host gene can have dramatic efects on
favivirus RNA synthesis. The identification of this host gene product and the
cell type itself, in which defective flavivirus RNAs can be readily generated
and propagated, could have tremendous value in future studies to determine
the cis-acting sequences involved in RNA replication and packaging.
COMPARATIVE ASPECTS
Genetic Diver
g
ence amon
g
Flaviviruses
Classification of flaviviruses has traditionally relied on serological tests.
Amino ac;id-sequence data can now be used to compare the relatedness of
different flaviviruses and the results of such comparisons are in general
agreement with those derived fom cross-neutralization tests with polyclonal
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678 CHAMBERS ET AL
DEN2

DEN3
N f
DEN4
a
JE 49 45 47 4
MV 48 45 46 46
1
W 49 47 45 47
B
YF 41 42 39 37 43 42 4
TBE 36 35 36 36 36 38 39 38
Z
C o =

<
f
<
<

U
Q Q Q ~ :; >
Figure 1 1 Homology matrix between different favivirus E proteins. Pairwise comparisons of
aligned E sequences (% identical residues). Serological subgroup clusters are indicated by
underlined bold type.
antisera (4, 99, 1 00, 1 24, 134, 166). Figure 1 1 compares the E protein
sequence homology (% identical residues) between several different flavivir
uses. From these data, the subgroup relationships are apparent; the DEN
serotypes cluster, as do the members of the JE subgroup; YF is distantly
related to these groups; TBE shows similar divergence from each of the
mosquito-bore virus groups, but examination of aligned NSI sequences
suggests that TBE is somewhat closer to YF ( 1 00) .
Oligonucleotide fngerprinting or restriction site polymorphisms have been
used as epidemiological tools to study the genetic divergence of independent
isolates of particular viruses difering in their geographic location, time of
isolation, animal source, and in some cases their virulence phenotype. Studies
with DENI ( 1 30) , DEN2 ( 1 63, 1 64) , DEN4 (72) , MVE (93, 96), SLE ( 1 65),
and YF (36) revealed a remarkable genetic homogeneity, although strain
differentiation can be detected and in general correlates with geographic
origin. More recently, limited nucleotide sequence comparison of MVE (94) ,
DENI (23), and DEN2 (6) isolates yielded similar results. Much of the
genetic stability observed for faviviruses in nature probably refects their host
adaptation and the selection against alterative genotypes.
Researchers hoped that such comparisons, together with epidemiological
and clinical data on disease severity, might allow the definition of specifc
genetic markers for virulence. Thus far, no clear picture has emerged. An
other approach to this question involves the comparison of attenuated vaccine
strains and their virulent parents. The sequences of attenuated strains of YF
(57) and JE ( 120) have been compared to their virulent parental stains. Both
attenuated strains differ not only in their virulence in primates but also in their
ability to be transmitted by mosquitoes. Comparisons identifed a number of
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FA VIVIRUS GENOME STRUCTURE & EXPRESSION 679
nucleotide and amino acid changes distributed throughout the genomes. For
example, the YF 1 7D vaccine strain and its virulent parental strain, Asibi,
which were separated by about 240 serial passages, differed at 67 nucleotide
positions (57, 1 32), leading to 32 amino acid substitutions. Given the ability
to recover infectious virus from cloned cDNA ( 1 32), it should now be
possible to determine which of these changes are biologically signifcante
Similarities to Other Positive-Strand Viruses
Comparisons of nucleotide and amino acid sequences (reviewed in 1 53, 1 56)
and high resolution structure determination of animal and plant viruses have
revealed common themes as well as a diverse spectm of genome organiza
tions and strategies for replication. As discussed above (see also 3) , virus
specifed proteinases are common to many positive-strand virus families, as
are sequence motifs suggestive of NTP-binding domains, helicase enzymes,
and RNA- dependent RNA polymerase activities. Although faviviruses were
originally grouped with alphaviruses based on their similar morphologies and
modes of transmission, in terms of genome organization and expression, the
flaviviruses clearly use a strategy similar to the picoma-like viruses (88) . Both
viral genomes encode a single polyprotein that begins with the structural
proteins and is proteolytic ally processed to yield the mature viral proteins. In
contrast, virion morphogenesis and strctural protein processing of favivir
uses are strkingly similar to rubella virs (42, 73), which makes up a separate
genus in the togavirus family along with the alphaviruses (see also 143). Both
viruses probably utilize membrane-anchored precursor forms of the C protein
and acquire their envelopes by budding through intracellular membranes.
Most closely related to the faviviruses, however, are the pestiviruses, also a
genus of the togavirus family. Recent genome sequences for bovine viral
diarhea virus (BVDV) (30) and hog cholera virus (HCV) ( 1 08) suggest a
similar genome arrangement and replication strategy to faviviruses (29).
However, scant amino acid-sequence homology exists between the pestivir
uses and the flaviviruses, and homology is primarily found in the motifs
discussed above for NS3 and NS5. The recently identified agent of non-A,
non-B blood-bore viral hepatitis (hepatitis C virus) (22) also shares similar
limited homologies with the flaviviruses and pestiviruses (C. Rice & S.
Feinstone" unpublished data) (Figure 8).
CONCLUDING REMARKS AND FUTURE PROSPECTS
Molecular studies on flaviviruses have entered an explosive phase. The past
several years have seen an amazing accumulation of structural information,
and common as well as distinct features of flavivirus genome RAs and
proteins have begun to emerge. However, for nearly every step of the virus
life cycle major gaps in our understanding still exist. Early events in virus-
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680 CHAMBERS ET AL
host interaction such as receptor binding, penetration, and uncoating are still
poorly understood. Substantial progress has been made in understanding viral
polyprotein processing. In contrast, little is known of the molecular in
teractions important for virion formation and the assembly and function of the
membrane-associated RNA replication complexes. The construction of cDNA
clones capable of producing infectious RNA tanscripts ( 1 32) should facilitate
a directed molecular genetic approach for studying many of these areas of
favivirus biology. Site-directed mutagenesis and constuction of recombinant
viruses with homologous or heterologous substitutions will allow stucture
function analysis of viral proteins and RNA sequences, and genetic mapping
of any interesting biological phenotype including changes important for
attenuation in vertebrate hosts and for arthropod vector competence. Func
tional cDNA clones can also be used for expression of individual viral
proteins or production of RNA substates for the development of in vito
enzymatic or RNA binding assays. Many other avenues are worth pursuing.
Among these, the development of in vitro RNA replication systems capable of
utilizing exogenous template RNAs would be invaluable for studying early
events in RNA replication. Ultimately, a high resolution three-dimensional
structure of a favivirus E protein and/or virion will be helpful in understand
ing virion assembly and virus-host interactions. One hopes that continuing
advances in our basic knowledge of favivirus biology will eventually lead to
more efective methods for contolling this important group of pathogens.
ACKNOWLEDMENTS
We thank L. Dalgamo, S. London, and M. MacDonald for many helpful
comments on the manuscript; C. Mandl, F. Heinz, and D. Trent for providing
manuscripts prior to publication; the Pew Memorial Trust, USAMRIID, the
Rockefeller Foundation, and NIH for fnancial support. C. S. Hahn is a
Johnson and Johnson Fellow of the LSRF. We apologize to our colleagues for
the omission of certain subjects and the incomplete literature citations because
of space limitations.
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682 CHAMBERS ET AL
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