DNA and RNA viruses Replication

Presenter: Dr. Arwa Dr. Jonathan Dr. Jannet Facilitator: Dr, Solomon

Outline
Definition of virus Characteristics of viruses Classification of the viruses Structure of the virus Virus replication

Definition
Virus is a sub cellular organism with a parasitic intracellular life cycle. it contains either DNA or RNA but not both. Outside the host cell a virus is not actually alive.

 What makes a virus? It contains nucleic acid genome surrounded by a shell of proteins(it may also contain lipids and sugars). Basic function is to deliver the viral genome into a cell where it can replicate.

General characteristics of virus

Are infectious agents with non living and living characteristics. They infect animals, plants and even other microorganisms. Viruses that only infect bacteria are called bacteriophages. Viruses that only infect fungi are called mycophages.

Living characteristics of viruses

They reproduce only in living host cells. They are composed of the same basic compounds as other forms of life( proteins, sugars, DNA/RNA and lipids). They can replicate in living organisms. They can mutate.

Non living characteristics of viruses .

They are acellular, that is, they contain no cytoplasm or cellular organelles. They carry out no metabolism on their own and must replicate using the host cell metabolic machinery. The vast majority of viruses possess either DNA or RNA but not both.

Virus classification
The formal taxonomical system for the classification of viruses is administered by the International committee for the taxonomy of Viruses is based on several structural properties. 1. Size : parvovirus is 20nm,poxvirus 450*250 nm. 2. Nucleocapsid symmetry (helical,icosahedral or complex)

 3.Presence of an envelope membrane (originally defined as either resistant or sensitive since the organic solvent will destroy the envelope membrane). 4. Type of genome (DNA or RNA). Viruses are classified into families (ending viridae), subfamilies(ending-virinae) and genera (ending -virus) on the bases of these criteria.

Dg virion

Baltimore classification system..

It classifies different viruses based on their unique replication strategies. They are seven different replication strategies based on this system. CLASS 1: Double stranded DNA viruses e.g herpesviridae, Adenoviridae, Poxviridae, Papovaviridae. These are among the largest of viral genome. CLASS 2: Single stranded DNA viruses e.g Parvoviridae, Circoviridae. These are typically small genomes.

 CLASS 3: Double stranded RNA viruses: e.g Reonaviridae,Birnaviridae. As with all RNA genomes these tend to be smaller than most DNA genomes. Genome consisting of between two and 12 different molecules of RNA (segmented genome). CLASS 4:single stranded RNA viruses- positive sense: eg Coronaviridae, Flaviviridae and Picornaviridae. CLASS 5: Single stranded RNA viruses negative sense e.g Orthomyxoviridae,Paramyxoviridae, Rhabdoviridae, Filoviridae and Togoviridae.

 CLASS 6: Positive sense single stranded RNA viruses that replicate through a DNA intermedite. E.g Retroviruses CLASS 7: Double stranded DNA viruses that replicate through a single stranded RNA intermediate. e.g. Hepadnaeviridae.

Viral replication

Is the term used by the virologist to describe the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. For it to enter it must have receptors which can bind to the target cells. Viral replication allow production and survival of its kind by generating abundant copy of its genome and capable of infecting new host.

 Replication between viruses is greatly varried and depends on the type of genes involved in them. Most DNA viruses assemble in nucleus while most RNA viruses develop solely in cytoplasm.

DNA virus replication

Viruses with dsDNA genomes. Replication occurs in the nucleus after viral entry. Some require host cell polymerases to replicate their genome while others like Herpesviridae and Adenoviridae encode their own replication factors. They produce different group of proteins at particular and appropriate time.

Herpes simplex virus

The virus enters the cell the protein contained in the viral tegument and capsid alters the cellular function and allow the synthesis of the first batch of viral messenger RNA and proteins. These are referred as immediate-early or alpha proteins, mainly regulatory. They allow the synthesis of the next set of protein(early or beta protein). Most such proteins are enzyme concerned with the synthesis of viral DNA and also preparing the cell for the manufacturer of the virus.

 Early proteins together with the newly synthesized viral DNA allow the synthesis of the late or gamma proteins(these are the structural component of the new viruses). DNA synthesis require short (usually RNA) primers. The basic mechanism of replication for Herpes virus involves circularization of the linear Herpes virus genome.

 Viral ds DNA genomes are than rolled off as very long polymers of the genome(concatamers) which are cut to unit length during viral assembly. With the Herpes virus the viral mRNA must go to the cytoplasm to be translated to proteins and then return to the capsid around the herpesvirus genome. Capsid assembled in the nucleus bud through the nuclear membrane transported to the cell surface in the cellular vacuole via exocytosis.

ssDNA virus replication ..

Example : Parvoviridae. To enter host cells, parvoviruses bind to a sialic acid -bearing cell surface receptor. Penetration into the cytoplasm is mediated by a phospholipase A2 activity carried on the amino-terminal peptide of the capsid polypeptide. Once in the cytoplasm, the intact virus is translocated to the nucleus prior to uncoating. Transcription only initiates when the host cell enters S-phase under its own cell cycle control, at which time the cell's replication machinery converts the incoming single strand into a duplex transcription template, allowing synthesis of mRNAs encoding the non-structural proteins, NS1 and NS2.

 The mRNAs are transported out of the nucleus into the cytoplasm where the host ribosomes translate them into viral proteins. Viral DNA replication proceeds through a series of monomeric and concatemeric duplex intermediates by a unidirectional stranddisplacement mechanism that is mediated by components of the cellular fork, aided and orchestrated by the viral NS1 polypeptide. Mature virions may be released from infected cells prior to cell lysis, which promotes rapid transmission of the virus, but if this fails then the virus is released at cell lysis.

Double stranded DNA viruses that replicate through a single stranded RNA intermediate.
e.g. Hepadnaeviridae(hepatitis B) This small group of viruses have a double stranded, gapped genome that is subsequently filled in to form a covalently closed cycle that serves as a template for production of viral mRNAs and subgenomic RNA.

 Entry in the target cell is mediated by IgA receptor on the cell surface. Once within the target cell the genome is converted to full double stranded form by virion enzyme. Following this a full length positive sense mRNA is synthesized along side multiple overlapping mRNAs. Atleast 3 types of mRNA are produced. The mRNA are translated to produce the four known Hepatitis B proteins which are C(core),P(polymerase/reverse transcriptase),S(surface) and X(transactivator)

 The viral P protein copies the full length RNA into a full length negative sense DNA. The positive sense DNA strand is transcribed from this DNA. The function of the X protein is not yet clear although it appears to transactivate some cellular genes and may involve in the induction of hepatocellular protein. A capsid of C protein forms around the genome.

RNA viruses replication

As with DNA,RNA is referred to as positive sense (containing the mRNA sequence) or negative sense (complementary to the mRNA sequence). Viruses with RNA genomes have smaller genomes thsan DNA viruses. The largest vertebrate virus RNA genome is about 1/10th of the size of the largest DNA genome. (no proofreading) Viral RNA genomes may be double or single stranded. Single stranded can be positive sense or negative sense or both.

Double stranded RNA virus replication

Reovirus have been studied extensively and have provided the model for dsRNA ceviruses in general. After entering the cell, reoviruses are digested by endosomes(they require low pH digestion step to infect the cell). Some are released by unknown mechanism and some remain in the lysosomes. The digested form is transcriptionally active form. RNA are made in the cytoplasm within the core.

 Only negative sense are trascribed producing positive sense mRNA which are released from the core while the parental RNA remains inside. The released of mRNA are then used both as mRNAs for protein synthesis and as virion components that are encapsidated into virion percusors. Once within the new virus,a single round of negative sense RNA ncopies are made from the mRNA, and these are yet used to produce more mRNAs.

 The genomic positive RNA strand is actually is an a with mRNA, complete a 5 cap modification seen exclusivelyon eukaryotic mRNAs. While the cap of an mRNA is involved with the forming the initiation complex for translation to protein, reoviruses alter biochemistry so that uncapped mRNAs are preferrentially translated late in infection, at the expence of capped mRNAs. Since all cellular mRNAs are capped this turns off cellular protein synthesis.

 However, the mRNAs produced from progeny reoviruses have no cap structure, and are translated preferentially. Reovirus are very efficient at shutting off cellular synthesis, and specifically inhibit DNA, RNA and protein synthesis. Mature reovirus is released by cell lysis.

ss RNA viruses replication

Positive sense Is an infectious in itself since it can code for production of viral protein by functioning as mRNA. E.g polio virus Since the genome can act as a mRNA there is no need for the virus particle to contain an RNA dependent RNA polymerase.

 The polio virus genomic RNA is translated to produce a single protein of more than 200kDa. While it lacks a cap, other regions in RNA (and possibly the viral VPg protein which is attached to the end of the genomic RNA molecule) seem to allow ribosome binding. This polypeptide is then repeatedly cleaved to produce not only the structural proteins that make up the virion, but also all of the enzymes necessary for replication,the polymerase.

 The polymerase then produces the full length nregative sense RNAs,which in turn are copied into positive sense RNAs. Some of the positive sense are translated, while others are packed as viral genomes by the newly synthesized coat proteins(which are still being cleaved even as the assembly occurs) . Assembled polio virus exits by lysing the cell.

 Other viruses with positive sense ssRNA genomes may produce more than one mRNA, allowing greater control of the production of individual proteins, and some exihibit limited temporal control, with early (replication) and late (structural) proteins being produced at different times in the replicative cycle.

Viruses with negative strand ssRNA genomes

ssRNA genomes complementary to mRNA can not function as mRNAs and require a polymerase to produce mRNAs before any viral use of the cellular synthetic machinery can occur. Due to lack of complexity inherent in their small size most RNA viruses do not have complex control mechanisms.

 Influenza virus, the virus requires cellular RNA synthesis since it can not make the 5 cap structure. It has a special function which removes caps from cellular mRNA and incorporates them into viral mRNA. Unusually for a virus with an RNA genome, it requires the viral mRNA to synthesized in the nucleus to allow capping. Followed by transport to the cytoplasm for translation. The influenza virus polymerase is also required to make full length copies of the genome which are inturn used to make progeny genomes.

 Influenza virus like other viruses with small genomes, some of the gene segments code for multiple proteins using overlapping reading frames and splicing of mRNAs. Unlike other ssRNA viruses of vertebrates influenza has a problem of its segmental genomes. For influenza virus to be infectious it should have all the genome segments (8). If infected by more than one virion chances for successful infection increases.

 some groups of viruses have ambisense genomes (some regions positive sense, others negative sense). These include the Arenaviridae and the Bunyaviridae, and appear to replicate in a manner broadly similar to the negative strand viruses, using a viral polymerase to make mRNAs rather than having a genome that is directly translated.