Luban-1

Eukaryotic Molecular Biology 2000

Jeremy Luban
Departments of Microbiology and Medicine
HHSC 1502
305-8710
http://cpmcnet.columbia.edu/dept/luban_lab/

RETROVIRUSES

Lecture Topics (3/27 and 3/29):

1. Significance of Retroviruses
2. Overview of the Retrovirus Life Cycle
3. Structure and Transformations of the Retroviral Genome
4. Virion Structure
5. Viral Entry
6. Mechanism of Reverse Transcription
7. Mechanism of Integration
8. Transcriptional Regulation
9. Post-Transcriptional Regulation
10. Virion Assembly
11. RNA packaging
12. Consequences of Retroviral Integration
13. Oncogene Transduction
14. LINES and other Retrotransposons
15. Pseudogenes: Transposition of a Reverse Transcript

Papers for Recitation (3/31):

1. Mann, R. et al. 1983. Construction of a retrovirus packaging mutant and its
use to produce helper-free defective retrovirus. Cell 33:153-159.
2. Boeke, J. D. et al. 1985. Ty elements transpose through an RNA
intermediate. Cell 40:491-500.

Review Articles:
1. Varmus, H. 1988. Retroviruses. Science 240:1427-1435.
2. Coffin, J. M. 1996. Retroviridae: the viruses and their replication, p. 1767-
1847. In Fields, B. N., et al. (3rd ed.), Fields Virology. Raven Press, Ltd.,
New York.
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I. Significance of Retroviruses

A. Among first known viruses: filterable, cancer-causing agents

B. The identification of reverse transcription provided an exception to

the Central Dogma.

C. Model for mobile genetic elements known as Retrotransposons

D. Cellular oncogenes were discovered as transforming activities

transduced by retroviruses.

E. Model systems for transcription, translation, recombination, and

other basic cellular processes.

F. Some of the most useful vectors for gene delivery to vertebrate cells.

G. Retroviruses cause diseases, including AIDS, cancer, and

neurologic syndromes.

II. The Retroviral Life Cycle: Overview

Retroviruses are replicating genetic elements that may be exogenous or

endogenous (carried within a cellular genome). Shown below is a
schematic diagram depicting the life cycle of an exogenous retrovirus.
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III. Transformations Undergone by the Retroviral Genome

A. The virion-associated genome consists of two copies of a capped

and polyadenylated RNA.

B. Genomic RNA serves as template for reverse transcription in

the cytoplasm of a newly infected cell. The reaction is catalyzed
by the viral reverse transcriptase and results in a linear double
stranded DNA molecule.

C. The DNA copy is transported to the nucleus where it

integrates into host chromosomal DNA, establishing the
provirus. The provirus becomes a permanent genetic
element of the cell, and is transcribed by the cellular RNA
polymerase II. The full-length RNA transcript serves as mRNA
and as genomic RNA that is packaged into newly synthesized
virions. It is also spliced to produce shorter mRNA species.

Genomic RNA (virion)

5'Cap R U5 Gag Pol Env U3 R An
Primer Binding Site Poly Purine Tract

Reverse Transcriptase

Double-stranded DNA (provirus)

U3 R U5 GAG POL ENV U3 R U5
5' LTR 3' LTR

RNA Polymerase II

Unspliced Transcript: gag and pol mRNA and nascent viral genome
SD SA
5'Cap R U5 Gag Pol Env U3 R An

Spliceosome

mRNA for env open reading frame

SD/SA
5'Cap R U5 Env U3 R An
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IV. Virion Structure

A. Membrane Envelope: taken from the producer cell plasma

membrane during virion production. Lined by the gag-encoded
Matrix protein.

B. Env-Encoded Glycoproteins:

1. TM (transmembrane): contains peptide that promotes

fusion of virion membrane with target cell membrane.

2. SU (surface): located outside the membrane, non-

covalently attached to TM. Binds to specific proteins on
the surface of cells (receptors), targeting these cells for
infection.

C. Virion Core: Round or triangular structure composed of gag-

encoded Capsid protein. Within the core are located:

1. two copies of viral genomic RNA, coated by the gag-

encoded Nucleocapsid protein

2. pol-encoded enzymes reverse transcriptase and integrase

Integrase Lipid Bilayer

Genomic
Matrix, p17
RNA

Reverse
Capsid, p24
Transcriptase
Nucleocapsid, p7
An An

Surface Transmembrane
glycoprotein (gp120) glycoprotein (gp41)
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V. Two Stages of Virion Entry (the example of HIV-1):

A. Binding: SU (gp120) on the surface of the virion binds to CD4, a

cell surface protein expressed on some T-cells and o n
macrophages. Binding to CD4 induces a change in the
conformation of SU which allows it to interact with a
chemokine receptor (CKR). The CKRs are a family of cell-surface
G-proteins which otherwise function as receptors for signaling
molecules called chemokines.

B. Membrane Fusion: The fusion peptide on gp41 is unmasked

when gp120 alters its conformation. The fusion peptide inserts
into the target cell membrane, bringing virion and target cell
membranes in proximity so that membrane fusion may occur.

Fusion
Peptide
CD4
gp120

CKR
gp41

Virion
Target
Membrane
Cell
Membrane

VI. Reverse Transcription: a complex series of reactions producing a

double-stranded DNA copy of the viral genomic RNA.

A. Occurs in the cytoplasm of the newly infected cell, within a

complex of viral proteins.

B. The DNA product has two identical Long Terminal Repeats

(LTRs), one at either end, making it longer than the RNA
template. This surprising result is due to two “jumps” carried
out by reverse transcriptase.

C. Components of the reaction include:

1. Template: viral genomic RNA

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2. Primer: a specific tRNA (taken from the producer cell during

viral assembly) that anneals to homologous sequences (primer
binding site or PBS) near the 5’ end of the viral RNA.

3. Reverse Transcriptase: a viral DNA polymerase that utilizes

RNA or DNA as template. It also possesses RNAseH activity that
degrades RNA when complexed with DNA.

3 ’ tRNA 5 ’
enomic (+) RNA
5’ R U5 PBS PPT U3 R 3’

STEP 1: tRNA extended

to form (-) strand strong
3’ R U5 tRNA 5 ’
stop DNA 5’ R U5 PBS PPT U3 R 3’

STEP 2: RNAse H
removes RNA hybridized
3’ R U5 tRNA 5 ’
to DNA 5 ’ PBS PPT U3 R 3’

STEP 3: The first jump:

(-) strand DNA hybridizes 5 ’ PBS PPT U3 R 3’
with remaining R sequences 3’ R U5 tRN A 5 ’

STEP 4: (-) strand DNA 5 ’ PBS PPT U3 R 3’

extended 3 ’ PBS PPT U3 R U5 tRN A 5 ’

STEP 5: Majority of 5 ’ PPT 3 ’

hybridized RNA
removed by RNaseH 3 ’ PBS PPT U 3 R U5 tRN A 5 ’

STEP 6: 3' end of

(+) DNA strand 5 ’ PPT U3 R U5 PBS 3 ’
extended 3 ’ PBS PPT U3 R U5 tRN A 5 ’

STEP 7: RNA and 5’ U3 R U5 PBS 3 ’

tRNA removed 3 ’ PBS PPT U 3 R U5 5’

STEP 8: The second 5’ U 3 R U5 PBS 3 ’

jump
3 ’ PBS PPT U3 R U5 5’

STEP 9: Both DNA 5’ U 3 R U5 PBS PPT U3 R U5 3’

strands completed
3’ U 3 R U5 PBS PPT U3 R U5 5’
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VII. Integration: covalent linkage of the double-stranded DNA copy of the

viral genome to host chromosomal DNA. The provirus is a permanent
genetic element in the infected cell, and in all of that cell’s progeny. A single
viral enzyme, Integrase, is required for integration:

A. Integrase is a nuclease which clips two nucleotides from the 3’

ends of the two LTRs, then:

B. In a concerted reaction, Integrase makes a staggered cut in host

chromosomal DNA, in a random location within the genome,
and ligates the clipped 3’ ends of the viral DNA to host DNA.
Host enzymes repair the 5’ ends of the viral DNA.

5'-LTR
...CATT-3’ 5’-AATG...
...GTAA-5’ 3’-TTAC...
3'-LTR

Cleavage of linear viral DNA tips

OH 5'-LTR
- 3’
...CA 5’-AATG...
...GTAA-5’ -AC...
OH
3'-LTR 3’

...5’-TTACCTTCATCTCTGTTCAG-3’... Random DNA

...3’-AATGGAAGTAGAGACAAGTC-5’... Target

Creation of staggered break in target DNA. Simultaneously,

the recessed 3’ ends of the viral DNA are joined to the 5’ ends
of the target.

5'- A
A
...5’-TTACCTTC-3’ TG... ...CAATCTCTGTTCAG-3’...
...3’-AATGGAAG TAGAAC... ...GT 3’-GACAAGTC-5’...
A
A -5'

Host enzymes remove unpaired bases,

fill gaps, and close nicks.

...5’-TTACCTTCATCTTG... ...CAATCTCTGTTCAG-3’...
...3’-AATGGAAGTAGAAC... ...GTTAGAGACAAGTC-5’...
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VIII. Proviral Transcriptional Regulation (HIV-1)

A. The U3 domain of the 5’ LTR of the provirus contains the single

retroviral promoter. The activity of this promoter is regulated i n
the same manner as other cellular promoters. For example, in
the case of HIV-1, it contains recognition sequences for the
transcriptional activator NF-kB. When cells are activated, NF-kB
translocates to the nucleus and activates the promoters of such
genes as IL-2. Similarly, it stimulates HIV-1 transcription. In this
way, HIV-1 expression responds to the activation state of the cell.

R U5
TATA

C/EBP NFkB/NFAT SP1

B. A viral protein called Tat stimulates transcription from the HIV-

1 LTR. In a cooperative fashion with cyclin T-CDK9, Tat binds to
TAR, an RNA stem-loop structure located at the beginning of
the viral transcript. CDK9, also know as P-TEFb (positive-acting
transcription elongation factor), phosphorylates the carboxy-
terminal domain (CTD) of RNA pol II, enhancing the
processivity of the polymerase.

RNA pol IIa

CDK9

Cyclin T
Tat P P P P
TAR RNA pol IIo

U3 R U5
Flanking chromosomal DNA
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IX. HIV-1 Post-Transcriptional Regulation

A. Despite the fact that HIV-1 has only one promoter which directs
synthesis of a single full-length RNA, more than 30 different
HIV-1 mRNAs are produced by alternative splicing using a
complicated array of splice-donors (SDs) and splice-acceptors
(SAs).

S S SS S RRE S
A A AA A A

VIF REV TAT

POL TAT ENV NEF
5’-CAP- R U5 GAG VPR VPU R U3 -A N -3’
REV
S S S S
D D D D

B. The genes encoding the major virion proteins, gag, pol, and
env, are translated from full-length or partially spliced RNAs
which possess multiple splice donors and acceptors. In order for
these RNAs to be translated, they must exit the nucleus without
being spliced. This requires direct interaction between a viral
protein, Rev, and a stem-loop structure in the unspliced RNAs
called the RRE (Rev-response element). The LPPLERLTL nuclear
export signal (NES) in Rev is recognized by a protein called
Exportin 1, which, as a complex with the RAN GTPase, targets
Rev with its associated RNA to the nuclear pore for export to the
cytoplasm.

Nuclear Membrane

RanGTP Exportin1 NES

REV

RRE
R U5 U3 R

SD SA
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X. HIV-1 Virion Assembly

A. The Gag polyprotein directs virion budding and release. It is

myristylated at the amino-terminus and accumulates at the
inner face of the cytoplasmic membrane where it multimerizes
to form the virion macromolecular complex.

B. The Gag-Pol polyprotein consists of the Gag polyprotein fused to

a large C-terminal extension containing the pol-encoded
enzymes, protease, reverse transcriptase, and integrase. In the
case of HIV-1 this protein is produced by a ribosomal frameshift
between the gag and pol reading frames. It is incorporated into
nascent virions via interactions with the Gag polyprotein.

C. The Envelope glycoprotein is an integral membrane protein

expressed in the secretory pathway. It is cleaved by a cellular
protease in the Golgi to produce TM, which spans the virion
membrane, and SU, which is noncovalently attached to TM, on
the outside of the virion.

D. Viral Genomic R N A is indistinguishable from full-length viral

mRNA. It is incorporated into virions via interaction with zinc-
fingers located in the nucleocapsid domain of the Gag
polyprotein.

Integrated Provirus
Nuclear Membrane
5'-LTR 3'-LTR

Plasma Membrane

Ψ
An
S D SA
An Budding Virion
Ψ
Ψ
:
SECRETORY PATHWAY

Env Polyprotein

CYTOPLASMIC
: Immature
Virion
Gag Polyprotein (contains trans packaging function)

Gag-Pol Polyprotein (results from ribosomal frameshift

or termination suppression).

Mature Virion
Results from
Activation of
Viral Protease
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E. Viral Protease Activation: When virions exit the cell, the Gag
and Gag-Pol polyproteins are intact, and the virion has an
“immature” morphology. Once the virion is released, the viral
protease is activated, the two polyproteins are cleaved, and the
virion attains a “mature” morphology (see section IV).

XI. RNA Packaging: Retroviral genomic RNA incorporation into virions

requires a cis-acting structure at the 5’ end of the RNA called Ψ and a
transacting factor, the zinc-fingers in the Gag polyprotein. Packaging
cell lines (see figure below) stably express all trans-acting factors
necessary for infectivity (gag, pol, and env), but no cis-acting factors. If
your favorite cDNA (YFcDNA) is fused to Ψ and introduced into the
cell line, virions will be produced capable of delivering your cDNA to
susceptible cell types.

Ψ+
YFcDNA Nuclear Membrane
5'-LTR 3'-LTR

Ψ− Plasma Membrane
gag pol env Ψ+ RNA
5'-LTR 3'-LTR

Ψ
Budding Virion
Ψ

Structural Proteins

YFcDNA
Packaged
into Virion
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XII. Consequences of Integration

A.
5'-LTR 3'-LTR

Insertional Mutagenesis:
integration of a replication
competent retrovirus alters
Exon 1 Exon 2 Exon 3
proto-oncogene transcription
c-onc

B.
c-onc
Oncogene Transduction: Exon 1 Exon 2 Exon 3
creation of transforming,
but usually replication
defective retrovirus.
5'-LTR 3'-LTR

Transducing viruses : Genes essential for viral replication are replaced by

cellular sequences. Replication usually requires the presence of a helper virus,
that provides the missing viral functions.

XIII. Transduction Mechanisms

A. Deletion

Proviral integration 5'-LTR 3'-LTR Exon 1 Exon 2

adjacent to a protooncogene c-onc
Deletion of viral 5'-LTR
and cellular sequences

Transcription

Processing An

Packaging of fusion RNA An

with viral genomic RNA An

Recombination during An
reverse transcription
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B. Readthrough

Proviral integration 5'-LTR 3'-LTR Exon 1 Exon 2

adjacent to a protooncogene c-onc

Exon 1 Exon 2
Readthrough
transcription SD SA

Processing An

Packaging of fusion RNA An

with viral genomic RNA An

Recombination during
An
reverse transcription

XIV. Retrotransposons are mobile genetic elements that replicate via reverse
transcription of an RNA intermediate. They can be divided into two classes:

A. Retrovirus-like (LTR) retrotransposons:

1. Ty elements are a family of dispersed, repetitive elements

in yeast. They resemble retroviruses with genes
corresponding to gag and pol, δ direct repeats at either end,
5 bp of target DNA repeated on either side.

2. Drosophila possess many transposable elements,

including 20-60 Copia elements which possess direct
terminal repeats, 5 bp direct repeat of target DNA, and
ORFs with gag and pol homologies.

B. Poly(A) elements lack LTRs and end with A-rich sequence.

1. Mitochondrial group II introns from S. cerevisiae. Target

site-primed reverse transcription begins at a site-specific
single-stranded break in recipient DNA.
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2. L1s (LINES, long interspersed nuclear elements)

a. 105 copies in all mammalian genomes (5% of genomic
DNA)
b. 97% are 5’ truncated (consistant with reverse
transcription mechanism)
c. polyA suggests that they arise from cDNA copies of
mRNA
d. Flanked by target site duplications, which presumably
result from staggered cuts in target DNA during
insertion
e. Integration sites are not sequence specific
f. reverse transcription and integration are probably linked
as shown below.
g. retrotranspositions are mutagenic and have resulted in
human diseases.

5' 3'
UTR ORF1 ORF2 UTR
EN RT An
L1 structure. 5’UTR (untranslated region) contains a promoter. ORF1 is an RNA-binding protein (like
gag) but it has no homology to any known protein. ORF2 has homology to RT and to endonucleases.

XV. Genesis of a Processed Pseudogene

Transcriptionally Exon 1 Exon 2 Exon 3

Active Gene

mRNA An

cDNA synthesis An
by unknown RT Tn

Transposition to location An
unrelated to site of origin Tn