Replication of DNA viruses

DNA Virus Replication strategies

General

· The virus needs to make mRNAs that can be translated into protein by the host cell translation machinery.

· The virus needs to replicate its genome.

· Host enzymes for mRNA synthesis and DNA replication are nuclear (except for Poxvirus in cytoplasm) and so, if a virus is to avail itself of these enzymes, it needs to enter the nucleus.

The DNA viruses can be split into 4 groups.

I: Papovavirus, Adenovirus and Herpesvirus (dsDNA viruses) - nucleus

Genomes are transcribed and replicated in the nucleus, and therefore can utilize the transcriptional enzymes of the host for generation of mRNA.

The transcription consists of at least two cycles for papovaviruses, and at least three for herpesviruses and adenoviruses. In each instance, the structural or virion polypeptides are made from mRNA generated from the last cycle of transcription.

II: Poxviruses (dsDNA viruses) - cytoplasm

Reproductive cycle takes place in the cytoplasm.

The genome is transcribed by a viral enzyme. The initial transcription occurs in the core of the virion.

III: Parvoviruses (ssDNA viruses)

The adeno-associated virus (human parvovirus), requires adenoviruses or herpes simplex viruses as helper viruses for its multiplication. In the absence of a helper virus, the genome appears to integrate into a specific locus of a human chromosome. Other human parvoviruses are capable of multiplying without the assistance of a "helper virus." Viral replication involves the synthesis of a DNA strand complementary to the single-stranded genomic DNA in the nucleus and the transcription of the genome.

IV: Hepadna viruses (dsDNA virus with RT e.g. Hepatitis B virus)

The DNA of this virus is first repaired and converted into a closed circular molecule by a DNA polymerase packaged in the virion, and then transcribed into two classes of RNA molecules, i.e. a mRNA specifying proteins and a genomic RNA which is transcribed by a reverse transcriptase to make the genomic DNA.

A) Type I: dsDNA

dsDNA viruses include:

• viruses of higher animals (Pox-, Herpes-, Adeno-, Papovaviridae)

Others:

• viruses infecting Eubacteria and Archaea, or phages (e.g. the Podoviridae, phages of E coli);
• viruses of insects (Baculo- and Irido- and Polydnaviridae);
• and viruses of eukaryotic algae (Phycodnaviridae);
• viruses of fungi

Size:

• 5-8 kb (Papovaviridae)
• through 30-40 kb (lambda phage and Adenoviridae)
• to about 200 kb (Baculoviridae)
• to over 300 kb (Herpes- and Pox- and Irido- and Phycodnaviridae).

Genomes:

• circular genomes (Papova- and Baculo- and Polydnaviridae);
• linear genomes (Adeno- and Herpesviridae, some phages);
• have circularly permuted linear genomes (phage T4, some Iridoviridae);
• or linear genomes with covalently closed ends (Pox- and Phycodnaviridae).

Replication of the viruses is in all cases by the semi-conservative method favored by cellular genomes; however, smaller circular genomes (e.g. Papovaviridae) replicate by means of bidirectional replication forks from a single origin, like some plasmids.

Among the viruses of Eukarya, replication mainly occurs in the nucleus, using cellular enzymes such as polymerases, methylases, etc. However, the replication of Poxviruses, some Baculoviruses (granulosis group), and some of the replication of iridoviruses, takes place in virus-specified "inclusion bodies" in the cytoplasm, using viral-coded enzymes, most important of which are DNA-dependent DNA polymerases.

B. Type II: ssDNA

ssDNA viruses include organisms infecting:

• bacteria ("bacteriophages", eg. Inoviridae, Microviridae)
• mammals (Circoviridae, Parvoviridae)
• birds (circovirus-like organisms),
• plants (Geminiviridae, banana bunchy top-like viruses or Nanoviruses).

Genomes:

• linear single-component genomes (Parvoviridae),
• circular single-component genomes (Microviridae, Inoviridae, Circoviridae, some Geminiviridae),
• circular two-component genomes (some Geminiviridae),
• or circular multicomponent (>3) genomes (Nanoviruses).

Genome size:

• the Circoviridae  have genomes of about 3 kb
• Parvoviridae have genomes of 4-5 kb
• the Geminiviridae about 2.7 - 5.4 kb
• the Microviridae (including the famous phiX 174) about 4.5 kb
• the Inoviridae and the Nanoviruses about 5-6 kb

Replication of all of the viruses requires formation of a "replicative form" (RF) double- stranded DNA intermediate: this is formed soon after infection, almost certainly by the host cell DNA polymerases engaging in "repair" of the ssDNA.

Parvoviruses have an interesting strategy for replicating their genomes, which uses internal or self-complementarity of genome ends to get around the problem of how to replicate a linear DNA genome. A virus-specific process is required both to nick RF DNA, and to sequester newly-formed genomic ssDNA into assembling particles: in the case of Parvoviridae the first is done by a NS1 protein (which binds the new 5'-terminus resulting from the nick) and the second by the coat protein; in Geminiviridae it appears as if the first is done by a similarly-acting  Rep protein, and the second also by the coat protein.

Steps in Replication

1. Conversion into dsDNA (=host repair process?)

2. Early transcription (by host enzymes)

3. Translation of (regulatory) protein and "rolling circle" ssDNA replication

4. Late transcription (usually mediated by viral proteins)

5. Synthesis of late (=structural) proteins

6. "Sequestering" of viral genomic ssDNA

7. Assembly into virions

Reference:

Parvoviridae: Fields Virology (2nd Edn), Chapter 62

Virus Taxonomy: Sixth report of the International Committee on Taxonomy of Viruses (FA Murphy et al., Eds.); Springer-Verlag, Wien, 1995.

Replication of Herpes simplex virus

· Larger virions (180 - 200nm) than adenoviruses

· Larger genome (90 -145x10⁶) than adenoviruses

· Linear, double-stranded DNA

· Enveloped icosahedral virus

Strategies:

Steps:

1) Attachment: HSV virion attaches to host cell with the envelope glycoprotein (gC, gB)

onto heparan sulfate moieties of cellular proteoglycans. Viral gD is believed to bind to

a secondary cellular receptor.

(2) Fusion and entry: The viral envelope fuses to the plasma membrane in a pH-

independent fashion such that the nucleocapsid enters the cytoplasm. gB, gD,

and gH are instrumental glycoproteins for this phenomenon.

(3) Uncoating: The capsid travels along the microtubules to a nuclear pore where the viral

DNA is released. The linear genome enters the nucleus and circularizes.

(4) Transcription: Once in the nucleus, the viral DNA is transcribed into mRNA by

cellular RNA polymerase II. In herpesviruses, viral gene expression is tightly

regulated and divided into 3 kinetic classes of expression.

I. A tegument protein associates with 2 cellular proteins, and the complex

transactivates transcription of HSV's five immediate-early (IE or alpha) genes.

IE genes generally encode regulatory proteins.

Alpha genes

There are five α genes which have been identified and described as ICPs (infected cell proteins), these include ICP0, ICP4, ICP22, ICP27 and ICP47. The α genes are by definition expressed in the absence of viral protein synthesis and contain the sequence GyATGnTAATGArATTCyTTGnGGG upstream of their coding regions. Their peak synthesis occurs 2-4 hours post infection, but they continue to accumulate until late in infection. All alpha genes appear to function as regulatory proteins with the possible exception of ICP47.

II. An IE protein initiates transcription of the early (E or β) genes. These gene

products are enzymes needed to increase the pool of nucleotides and for viral

replication.

Beta genes

These genes are not expressed in the absence of α proteins and their expression is enhanced in the presence of drugs which block DNA synthesis. They reach peak rates of synthesis 5-7 hr post infection. The genes have been subdivided into the β 1 and β 2 subclasses. β 1 genes appear early after infection, but require the presence of α 4 protein for their synthesis. Examples of β 1 genes include the large component of ribonucleotide reductase and the major DNA binding protein (ICP8). β 2 genes include viral thymidine kinase (TK) and the viral DNA polymerase. β gene synthesis immediately precedes the onset of viral DNA synthesis and most viral genes involved in viral nucleic acid metabolism appear to be β genes.

III. Late (L or γ) genes are activated for production of viral structural proteins.

Gamma genes

This class of genes is also separated into two groups. γ 1 genes are expressed early in infection and are only minimally affected by inhibitors of DNA synthesis (example, major capsid protein). In contrast, γ 2 genes are expressed late in infection and are not expressed in the presence of inhibitors of viral DNA synthesis.

The location of the gene classes within the HSV genome is of interest. α genes map at the termini of the long and short components and tend to cluster together. In particular, α genes surround the HSV origin of replication in the short region (oris). Each α gene has its own promoter-regulatory region and transcription initiation and termination sites. β and γ genes are scattered in both the long and short components. Interestingly, the β genes specifying the DNA polymerase and the DNA binding protein flank the origin of replication in the long region (oriL). There is little gene overlap and few instances of gene splicing for any of the HSV gene classes.

(5) Translation: After transcription in the nucleus, all mRNA transcripts are translated

into protein in the cytoplasm. Subsequently, the proteins can go to the nucleus, stay

in the cytoplasm, or become a part of the membrane bilayer.

(6) Assembly: Capsid proteins assemble in the nucleus to form empty capsids. Full-

length viral DNA is packaged to form nucleocapsids.

The nucleocapsids associate with segments of the nuclear membrane where tegument

and glycosylated envelope proteins have bound. This association triggers envelopment

by budding through the nuclear membrane.

Enveloped virions accumulate in the endoplasmic reticulum (ER).

(7) Release: Mature virions are released by exocytosis.

(8) Virus-specific proteins are also found on the plasma membrane of infected cells.

Functional requirements for viral replication

1. Proteins essential for viral origin dependent amplification

2. Enzymes involved in nucleic acid metabolism (thymidine kinase, ribonucleotide reductase, dUTPase, uracil DNA glycosylase, alkaline exonuclease.

Seven genes have been identified which are necessary for origin dependent replication: viral DNA polymerase, ICP8 (single stranded DNA binding protein), origin binding protein, dsDNA binding protein, and three other proteins which may be involved in primase and helicase activities.

The importance of the virally encoded enzymes in HSV replication is detailed below.

· Alkaline DNase: essential for viral growth and DNA replication

· Thymidine kinase (TK): broad substrate specificity

· Ribonucleotide reductase: reduces ribonucleotides to deoxyribonucleotides creating a pool of substrates for DNA synthesis. It is comprised of two subunits 140kd and 38kd. Both subunits are required for activity.

· Uracil DNA glycosylase: involved in DNA repair and proof reading, corrects insertion of dUTP into DNA.

· dUTPase: converts dUTP to dUMP preventing dUTP incorporation into DNA and providing a pool of dUMP for conversion to dTMP.

Regulation of viral gene expression

RNA polymerase II is responsible for viral mRNA synthesis. In general viral mRNAs are capped and polyadenylated just like cellular mRNAs. Only a small portion are spliced. DNA sequences upstream of HSV genes determine their capacity to be expressed as α or β genes. α gene expression is directed by α-TIF (VP16, vmw65) which complexes with cellular proteins to bind a TAATGArATT motif. Despite controversy over the ability of ICP4 to bind DNA, it appears to be support for specific ICP4 binding sites upstream of the TK gene (beta gene) and of ICP4 itself.

In general expression of HSV genes appear to be controlled by three means:(a) cis-acting sites for both viral transacting factors and cellular factors. (b) trans-acting signal proteins specified by the virus and (c) viral and cellular factors involved in viral DNA synthesis and post-translational modification of viral proteins.

3 "top guns" for viral gene regulation

Alpha-TIF: alpha trans-inducing factor (also known as VP16 and vmw65) is a structural component of the HSV virion (located in the tegument). In conjunction with cellular proteins alpha-TIF binds the TAATGArATT motif upstream of alpha genes and induces their transcription. alpha-TIF does not bind DNA directly and there are about 500-1000 copies of alpha-TIF per virus particle.

ICP0: also known as IE110. It is an alpha gene which can promiscuously transactivate transfected genes. Its function in infected cells is not known. ICP0 deletions mutants are still viable in cell culture.

ICP4: also known as IE175. It is the major transactivator of HSV genes and is an essential gene product. ICP4 is autoregulatory and probably turns off its own synthesis and ICP0 synthesis as well. ICP4 functions as both a transactivator and a repressor and may be regulated by post-translational modifications, position of the DNA binding site or strength of binding to DNA.

HSV latent infection:

Latent infection occurs in neurons found in sensory and autonomic ganglia. Initiation of latent infection occurs as in lytic infection upto immediate early gene expression step. It is unclear if tegument proteins are transported into the nucleus.

However, when the latent infection is established, transcription is restricted such that a single pre-mRNA is produced from the associated transcript (LAT) promoter. Low-level or sporadic transcription of immediate early and early genes can occur but is not sufficient to initiate a productive infection.

The latency associated transcript initially synthesized is spliced, and a stable intron in the form of lariat (2 kb LAT) is produced in the nucleus. The spliced LAT mRNA is transported to the cytoplasm, where small open reading frame (ORF-0 & ORF-P) may be translated into proteins. The function of LAT RNAs and the expression of ORF as proteins are not well understood.

(For more reading: Annual Review of Biochemistry: Herpes simplex virus DNA replication, Vol. 66: 347-384, July 1997).

Replication of Adenovirus

· Non-enveloped, icosahedral viruses with fibers

· 70nm diameter

· The DNA is linear, double stranded, associated with virally coded, basic proteins in virion (unlike papilloma and polyoma viruses (papovaviruses), adenoviruses do not use cell histones to package virion DNA)

Strategies:

· Adenoviruses code for their own DNA polymerase and DNA packaging proteins.

· However, although adenoviruses code for their own DNA polymerase, they use host factors in addition to viral proteins for DNA replication, and they use host RNA polymerase and RNA modification systems and so nucleic acid synthesis needs to be in the nucleus.

• Adenoviruses are capable of lytic infection, chronic infection in humans (lymphoid cells), and oncogenic transformation in certain animal cells.

Steps:

Adsorption and Penetration

Adenoviruses usually infect epithelial cells. The fibers bind to a cell surface receptor and the virus is engulfed by endocytosis. The virus appears to be able to lyze endosomes.

Uncoating occurs in steps. DNA is released into the nucleus (probably at a nuclear pore).

Early phase

Early transcription: Adenovirus uses host cell RNA polymerase and early mRNAs are transcribed from scattered regions of both strands. Multiple promoters result in more flexible control. mRNAs are processed by host cell capping, methylation, polyadenylation and (sometimes) splicing enzyme systems, they are then exported to the cytoplasm and translated.

The early proteins include those which:

· are needed for transcription (E1A protein is needed for transcription of the other early genes; as a result these other genes are sometimes referred to as "delayed early" genes and E1A is referred to as an "immediate early" gene). 

· are needed for adenovirus DNA synthesis (includes DNA polymerase)

· alter expression of host cell genes. This includes genes whose products interfere with the host anti-viral response and/or interfere with cell cycle regulation

Gene expression of Adenovirus

Immediate early E1A (Can immortalize primary cells in vitro.)

Early E1B, E2A, E2B, E3, E4, some virion proteins

E1B does not transform cells on its own, but "co-operates" with E1A to stably transform cells.

E1A + E1B: necessary for full transformation and tumour formation in animals.

Late Late genes, mostly virion proteins

Late phase

DNA replication:

Adenovirus encodes its own DNA polymerase (which is one of the early proteins). The DNA is replicated by a strand displacement mechanism. There are no okazaki fragments, both strands are synthesized in a continuous fashion.

DNA polymerases cannot initiate synthesis de novo, they need a primer. In the case of adenovirus, the virally coded terminal protein (TP) acts as a primer. It is thus found covalently linked to the 5' end of all adenovirus DNA strands.

DNA replication

Late transcription:

The way in which late transcription is switched on is not well understood. Late mRNAs code predominantly for structural proteins and there is one major late promoter. The primary transcript is processed to generate various monocistronic mRNAs:

There are two types of cleavage of primary transcript:

i. to generate various 3' ends which are then polyadenylated

ii. for intron removal

It is not understood how this process is controlled such that the correct amounts of each mRNA are made. It seems that the virus makes more mRNAs and proteins than are needed for virion assembly, so precise control may not be necessary.

Assembly

Assembly of adenovirus particles occurs in the nucleus. DNA enters the particles after immature capsids are formed. The capsids then undergo a maturation process, after which the cells lyse and virions leak out.

More structural proteins are made than are needed and excess structural proteins accumulate in the nucleus where they form inclusion bodies.

Replication of Hepatitis B Virus

• A major human pathogen causing acute and chronic liver inflammation
• A member of hepadnaviridae
• Enveloped DNA virus
• Narrow host range

Features:

• The HBV replication involves reverse transcription of a pregenomic RNA (pgRNA) and its polymerase molecule is covalently attached to the dsDNA.

• During initiation of infection, rcDNA ----------à cccDNA

(RT and RNA removed)

cccDNA-à template for transcription of viral mRNAs

Pregenome ---à mRNA for synthesis of core protein and viral RT

• RT --à binds to 5’ end of its own mRNA template, and complex is packaged into nucleocapsids, where viral DNA synthesis occurs.

· The viral DNA polymerase (the P protein) plays a crucial role in HBV genome replication. Like retroviral reverse transcriptases, this enzyme possesses both RNA- and DNA-dependent polymerase activities and an RNase H activity that degrades the RNA moiety of RNA/DNA replication intermediates. 

• Once partially ds DNA produced, nucleocapsid can go a maturation process that facilitates acquisition of an outer envelope via budding into ER

Steps:

Phase I: Docking of HBV to the membrane of a permissive cell and entry into the cytoplasm/endosome.

The attachment/entry is a poorly understood process as there is no good model organism for the entry stage of the HBV infection.  

Viral replication is most efficient within the liver.

Phase II: Separation and Transportation of the capsid-bound HBV-DNA in the cell nucleus.

Following uncoating the nucleocapsid is transported to the nuclear membrane, where the host polymerases will complete the partially double stranded DNA by the generation of a closed, circular DNA form:  

Phase III: Separation of the HBV-DNA and DNA-conversion of the HBV-genome into a covalently closed circular DNA by the viral DNA-polymerase

1. Extension of the plus-strand DNA 

2. Removal of the P protein (polymerase protein is the RevT protein and is only referred to with a different name) and the RNA primer. 

3. Ligation of the resulting 3'OH and 5'PO4- ends 

The former step may be carried out by the viral polymerase, while the latter steps require host cell enzymes. 

There are no transcription factors (like TAT) found in the HBV.  

Phase IV: Synthesis of surface antigens as well as viral capsid- and polymerase protein

Integration of HBV DNA into the host cell genome is not required for HBV replication. HBV genome directs synthesis of pregenomic RNA and viral mRNAs. Cellular RNA polymerase II recognizes four different promoters: PreS1, PreS2, C, and X Transcription is influenced by two enhancer elements (EnhI and EnhII) that provide binding sites for cellular transcription factors, including factors that are specifically enriched in hepatic cells (e.g. Hnf1, 3, and 4).  

Thus, the two enhancer elements can regulate transcription levels without interacting with viral proteins. Because the enhancers do associate with proteins found in hepatic cells, the virus is predominantly found in the liver.  

DNA Polymerase

The viral DNA polymerase (the P protein) plays a crucial role in HBV genome replication. Like retroviral reverse transcriptases, this enzyme possesses both RNA- and DNA-dependent polymerase activities and an RNase H activity that degrades the RNA moiety of RNA/DNA replication intermediates. 

Phase V: Reconstitution of the virus capsid, the virus envelope and the infectious virus

Phase VI: Formation of membrane vesicles with infectious and non-infectious virus particles

Phase VII: Exocytosis

The budding phase is thought to be similar to exocytosis, but similarly to the entry/attachment phase, little is known about the exact process. 

Important point to note:

• The X protein is implicated in the development of hepatocellular carcinoma (liver cancer). It stimulates viral gene transcription. It may stimulate transcription indirectly via activation of cellular signalling pathways.

· The core protein packages the HBV genome in the virion. 

· Surface proteins: 3 different in-frame start codons are utilized within the S ORF. These proteins (PreS1, PreS2, and S) are involved in HBV envelope formation.  

· It is thought that the reason why the DNA is only partially double stranded, is because there is probably less steric hindrance for a partially dsDNA molecule over a fully dsDNA molecule