INFECTIOUS DISEASE

VIROLOGY -  CHAPTER  THREE   

DNA VIRUS REPLICATION STRATEGIES  

Dr Margaret Hunt
Professor Emerita
Department of Pathology, Microbiology and Immunology
University of South Carolina School of Medicine

with additions by:

Dr Dorian McIlroy
University of Nantes

TEACHING OBJECTIVES

Descriptive analysis of the replicative strategies employed by animal DNA viruses

Identification of virus prototypes associated with different DNA virus replication schemes

GENERAL

Viral genomes contain information which:

• ensures replication of viral genomes

• ensures packaging of genomes into virions

• alters the structure and/or function of the host cell to a greater or lesser degree

VIRAL STRATEGY

Viral strategy refers to the manner in which each virus carries out the above functions. Since a virus is an intracellular parasite, it has to operate within limits imposed by the host cell, or circumvent these limitations.

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 those in mitochondrion) and so, if a virus is to avail itself of these enzymes, it needs to enter the nucleus.

 

Human parvovirus B19
Image courtesy of Dr J-Y Sgro
Used with permission

NUCLEAR DNA VIRUSES  

PARVOVIRUS FAMILY

Parvoviruses are very small (18 to 25nm diameter), single stranded DNA viruses (parvum=small) (Figure 1a). They have an icosahedral capsid, and are non-enveloped. DNA replication occurs in the nucleus.

Many human parvoviruses are satellite viruses that require co-infection by another DNA virus – either an Adenovirus, or a Herpesvirus – in order to replicate. These Adeno-Associated viruses (AAV) have been developed as gene therapy vectors. On the other hand, the bocaviruses (which cause respiratory infections) and Parvovirus B-19 are capable of replication in the absence of a helper virus.

Human parvovirus B-19 replicates in dividing cells – primarily in erythrocyte progenitors in the bone marrow - and causes fifth disease (erythema infectiosum). This is usually a mild disease but the decreased production of red blood cells can be a problem in people with various types of severe hemolytic anemia.

Adsorption, penetration and uncoating

Parvovirus B-19 binds to the erythrocyte P-antigen, or globoside, which is also present o the surface of erythrocyte progenitors. Binding is followed by virion endocytosis mediated by the α5β1 integrin, which is expressed by erythrocyte progenitors, but not by mature red blood cells. The use of an integrin co-receptor ensures that the virus does not enter red blood cells, which, as they lack a nucleus, are not capable of supporting parvovirus B19 replication.

After entry, the virion escapes from the endolysosome, due to the action of the minor capsid protein, VP1. Parvovirus virions are then actively transported via the microtubular network to the vicinity of the nucleus. They are small enough to be imported into the nucleus through the nuclear pore complex, and uncoating occurs in the nucleus.

Gene expression and DNA replication

The single stranded DNA genome is first converted to double-stranded DNA by host cell enzymes. The inverted terminal repeat (ITR) sequences at each extremity of the viral genome fold back on themselves, so that the free 3' OH group at the end of the virus single-stranded genome can be used as a primer for the synthesis of the complementary DNA strand.

The double-stranded parvovirus B-19 genome contains a single promoter element that recruits cellular transcription factors and RNA polymerase. Alternative splicing generates mRNAs coding for the two capsid proteins (VP1 and VP2) and the non-structural protein NS1. These viral proteins are all synthesized in the cytoplasm, then imported into the nucleus.

NS1 is essential for replication of the virus genome – but it is not a DNA polymerase. Instead, NS1 acts as an origin recognition protein, which specifically binds to double-stranded virus DNA and allows host DNA polymerase to replicate viral DNA, generating many single-stranded copies of the parvovirus genome. Parvovirus B19 can only replicate in actively cycling cells, that express all of the cellular factors necessary for DNA replication.

Both (+) and (-) strand copies are generated, and both can either:

•  be converted to double-stranded DNA genomes, which can then be used as templates for virus gene transcription and intermediates for virus genome replication

• be incorporated into virions

Assembly and Release

Assembly occurs in the nucleus, and virions are released by cell lysis.

PAPILLOMAVIRUS FAMILY

The Papillomavirus family was formerly grouped with the Polyomavirus family into the Papovavirus family (PApilloma, POlyoma, simian VAcuolating virus 40) because members of both families have a similar structure. However, it is now clear that the two families have a very different replication strategy and so each group has now been given its own family status

Papillomaviruses (figure 1b) are difficult to grow in culture.  They will not be discussed further in this section (but see section of DNA tumor viruses).   

These include SV40 (Figure 2), BK, JC and polyoma viruses. All have a similar strategy for DNA replication.  They are small (~40nm diameter), icosahedral, non-enveloped viruses that replicate in the nucleus. Depending on the host cell, they can either transform the cell (go here) or replicate the virus and lyze the cell. 

LYTIC CYCLE

Attachment, penetration and uncoating

Viral capsid proteins interact with cell surface receptors and penetration is probably via endocytosis. Virions are transported to the nucleus and uncoated. DNA (and associated histones) enters nucleus, probably through a nuclear pore

Production of viral mRNAs and proteins

Gene expression is divided into early and late phases.

Early genes encode enzymes and regulatory proteins needed to start viral replication processes.

Late genes encode structural proteins, proteins needed for assembly of the mature virus.

Figure 3 Early gene expression
Note: - - - - indicates regions of the primary transcript which are removed in the alternatively processed mRNA. Modified from Fiers et al.,Nature 273:113

 
Figure 4 Late gene expression
Note: - - - - indicates regions of the primary transcript that are removed in the alternatively processed mRNA. Broad arrows indicate regions translated into protein
Modified from Fiers et al.,Nature 273:113

 

EARLY PHASE OF THE LYTIC CYCLE

Early gene expression (figure 3 and 6)

The early promoter is recognized by host RNA polymerase II (SV40 contains a strong enhancer). Post transcriptional RNA modification (capping, methylation, polyadenylation, splicing etc.) is carried out by host enzymes. The early transcript  (primary transcript) is made and then undergoes alternative processing, resulting in the mRNAs for the small T and large T antigens (these proteins have common amino-termini but different carboxy-termini).

The mRNAs are translated in the cytoplasm.

Note: Primary transcripts which can be processed and code for more than one protein are seen in several virus families and in the host cell.

LATE PHASE OF THE LYTIC CYCLE

By definition the late phase starts with the onset of viral genome replication.

DNA replication

SV40/polyoma DNA replication occurs in the nucleus. 

Large T antigen is needed for DNA replication. This binds to the origin of replication.

Polyoma viruses use the host cell DNA polymerase, which recognizes the viral origin of replication if the T antigen is present.

DNA replication is bidirectional (There are two replication forks per circular DNA genome and replication involves leading/lagging strands, Okazaki fragments, DNA ligase, etc.). This process of DNA replication is very similar to that which occurs in the host cell - which is not surprising as the virus is using mainly host machinery except for the involvement of the T antigen.

Host histones complex with the newly made DNA.

 

Late gene expression (figure 4 and 6)

Late mRNAs are made after DNA replication (a lot of newly made viral DNA is now available as template). Early mRNAs are still transcribed, but at a very much much lower rate.

T antigen is involved in controlling increased transcription from the late promoter and decreased transcription from early promoter. It also interacts with host proteins and changes the properties of the host cell, thus playing a role in cell transformation and tumor formation.

VP1, 2, and 3 are made from same primary transcript which undergoes differential splicing (figure 5). This results in the reading frame for VP1 being different from that for VP2 and VP3. Thus, one region of the DNA can code for two different amino acid sequences according to which reading frame is used. This is another way that viruses (and cells) can use a short stretch of DNA to code for more than one protein sequence.

 

ASSEMBLY

VP1, 2 and 3 mRNAs are translated in the cytoplasm, the proteins are transported to nucleus, and capsids assemble with DNA (and cell histones) inside the capsid. Large numbers of capsids accumulate in the nucleus and form inclusion bodies. Virions are released by cell lysis.

 

Figure 7b Adenovirus structure

Adenoviruses are double stranded, non-enveloped icosahedral DNA viruses with a linear genome (Figure 7 and 8)

LYTIC CYCLE

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) (figure 9).

Early phase

Early transcription: Adenovirus uses host cell RNA polymerase and early mRNAs are transcribed from scattered regions of both strands (figure 10). 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.

 

A  
B Figure 8 Models of the adenovirus virion. A: A 3-dimensional image reconstruction of the intact adenovirus particle viewed along an icosahedral 3-fold axis (© EMBL Virus Structure Resource). B: A stylized section of the adenovirus particle based on current understanding of its polypeptide components and DNA. No real section of the icosahedral virion would contain all the components. Virion constituents are designated by their polypeptide numbers with the exception of the terminal protein (TP). Adapted from Fields et al., Fundamental Virology (1996).
 

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

 Figure 9  Diagrammatic representation of the uptake and uncoating of adenovirus particles. Adapted from Zinsser Microbiology 20^th Ed. 
Figure 10
Transcription map of adenovirus. Early genes are shown in red. Black indicates late genes. Blue lines indicate DNA. Square brackets indicate the positions of promoters. Primary transcripts are made from each promoter and then undergo alternative splicing, the diagram above does not show the primary transcript. It only shows those regions present in the alternatively spliced products.
Missing regions indicate removal of introns. Adapted from Broker, T.R. In Processing of RNA. (Apirion, D ed) 181-212, 1984

 

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 (figure 11). 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.

 Figure 12 Processing of viral primary transcript

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 (figure 12). The primary transcript is processed to generate various monocistronic mRNAs (figure 12):

There are two types of cleavage of primary transcript:

• to generate various 3' ends which are then polyadenylated

• 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.

 Figure 13a  Herpes virus structure

Figure 13b Herpes simplex virus  © Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission). 

HERPESVIRUSES  

Herpesvriuses have linear double-stranded DNA and are both enveloped and icosahedral (Figure 13)

Figure 15 Fusion of membrane-bound virus with the plasma membrane

 

Adsorption and penetration

Many herpesviruses, including herpes simplex virus, can fuse directly with the cell plasma membrane (which results in partial uncoating) (figure 14). Such fusion with the plasma membrane has implications for both the virus and the host cell. Among these are:

• Since the fusion protein is active at physiological pH, if it is inserted into the host cell membrane during the virus growth cycle, the infected cell can potentially fuse with other cells and form syncytia.

• The viral membrane leaves a "footprint" in the cell plasma membrane and this is a possible clue that the cell is infected (figure 15)

Capsids are transported towards the  nucleus and the DNA passes into the nucleus (probably via nuclear pores).

Early phase

Early transcription (the mRNAs made during this phase are the alpha and beta mRNAs) (figure 16)

Herpes viruses use host RNA polymerase. However, a virion tegument protein (VP16) enters the nucleus upon infection and is important as part of the transcription factor complex recognized by the host RNA polymerase. The virus uses host mRNA modification enzymes.

Initially, alpha-mRNAs are transcribed.  These are the immediate early mRNAs and are exported to the cytoplasm and translated into alpha-proteins. The α-proteins translated in the cytoplasm are transported into nucleus where they enable the beta-promoters to be used by the host RNA polymerase (figure 16).

Beta-mRNAs are transcribed the by host RNA polymerase. (Beta-genes are still "early" since they are transcribed prior to DNA synthesis. Sometimes therefore, alpha-genes are called "immediate early" and beta-genes are called "early"). Beta proteins are involved in gene expression regulation. They decrease alpha-gene expression and are needed for gamma gene expression. They are also involved in various aspects of DNA synthesis; for example, herpes beta -genes code for a variety of proteins including:

• DNA polymerase

• DNA binding proteins

• thymidine kinase

• ribonucleotide reductase

SINCE THESE BETA PROTEINS ARE VIRALLY-CODED AND NOT HOST-CODED ENZYMES, THEY ARE POTENTIALLY WEAK LINKS IN THE VIRUS LIFE CYCLE AND THUS PROMISING TARGETS FOR VIRAL CHEMOTHERAPY

Figure 17 Possible genomic structures of herpes viruses

Late phase

DNA replication

Herpesviruses code for several proteins, in addition to the DNA polymerase, that are needed for DNA replication. The precise mechanism of DNA replication is not known. DNA replication is accompanied by a lot of recombination. The replicated DNA is present as long concatameric molecules (tandem repeats of the genome linked head-to-tail). These are cleaved to genome-size lengths when the DNA is packaged into the virion. 

Some herpesviruses (e.g. herpes simplex virus) have a genome structure in which two parts of the genome can invert relative to each other (figure 17), others do not. The significance of this is unclear.

Late transcription:

By definition, late transcription occurs after DNA replication. Gamma mRNAs are made and are translated in the cytoplasm. Gamma proteins are predominantly structural. There is decreased expression of beta genes in the late stage. This is probably due to down-regulation of transcription of beta genes by one of the gamma proteins.

In herpesviruses there is no apparent organization of the genome into blocks for either early or late transcription.

Figure 18B
Herpes simplex virus on and in a peripheral blood lymphocyte  © Dennis Kunkel Microscopy, Inc.  Used with permission
 

Assembly

Assembly occurs in the nucleus. A capsid is formed and the DNA enters the capsid. The capsids acquire an envelope by budding through areas of the inner nuclear membrane which have viral membrane proteins inserted into them (figure 18). These areas have tegument proteins associated with the inner face of the inner nuclear membrane. The virus envelope then fuses with the outer nuclear membrane and the de-enveloped nucleocapisid is delivered into the cytoplasm, where it acquires a more mature tegument. It then becomes re-enveloped by budding into Golgi-derived vesicles and is then released.

The late protein required for transcription of immediate early mRNAs in the next round of infection is packaged in the virion.

  Excess structural proteins accumulate in nucleus, often form inclusion bodies (part of the cytopathic effect).

FEATURES TO NOTE ABOUT HERPESVIRUSES

There are no obvious blocks of early or late genes

They are more independent than some of the smaller viruses

Since they are more independent, there are more "weak links" that may be targeted by drugs

  Figure 19 Negative stain and thin section of pox viruses  © F. Fenner

CYTOPLASMIC DNA VIRUSES  

POXVIRUSES  

There are several reasons why poxviruses (figure 19) are of importance:

• Certain poxviruses are of historic note,  such as smallpox (figure 20) and vaccinia (cow pox, which was used in the smallpox vaccine (go here))

• Pox viruses may be possible agents of bioterrorism

• Pox viruses are used in new techniques of vaccine development (such as genetically-engineered vaccinia)
• Some members of this family infect man (molluscum contagium (figure 21), orf, monkey pox, cow pox). Note: chicken pox is caused by a herpes virus which  is not a member of the poxviridae

Poxviruses replicate in the cytoplasm. This means that they must provide their own mRNA and DNA synthetic machinery.

Vaccinia is the most intensively studied member of the poxvirus family.

Adsorption and penetration

The virus binds to cell surface receptors. It enters cells via endocytosis or by direct fusion of the virus with the plasma membrane. The virus is then released into the cytoplasm, minus its membrane.

 

Early phase

Early transcription

After the initial phase of uncoating has occurred, the virus can make a limited number of mRNAs (the immediate early mRNAs). To do this, the poxvirus needs a DNA-dependent RNA polymerase. Host RNA polymerase is in the cell nucleus and so this explains why poxviruses use a virally-coded DNA-dependent RNA polymerase to make their RNAs. Since this enzyme is needed immediately upon infection, it must be brought into the infected cell with the vaccinia DNA, it is thus present in virions. "Naked" vaccinia DNA which has had all the protein removed is thus not infectious, since it will have no RNA polymerase associated with it, and nothing can happen in the vaccinia life cycle without the vaccinia RNA polymerase since no RNA or proteins can be made.

Poxvirus mRNAs are capped, methylated and polyadenylated just like standard eucaryotic mRNAs, but host cell mRNAs are modified in the nucleus and vaccinia replicates in the cytoplasm. Since Vaccinia is cytoplasmic, these modifications must be carried out by virally-coded enzymes. The modifying enzymes are packaged in virions and thus those mRNAs made immediately upon infection can be modified. So far, no spliced mRNAs have been reported for vaccinia (this is not surprising since it replicates in cytoplasm and host splicing enzymes are in the nucleus).

One of the immediate early mRNA translation products is an uncoating enzyme. This allows further uncoating of the vaccinia DNA and more genes can now be transcribed - the early genes are now all expressed. Poxviruses are exceptional in that they code for an uncoating protein which has to be made in the newly infected cell before uncoating can be completed.

Virus production "factories" are seen in the cytoplasm - sites of vaccinia virus replication.

The early proteins are involved in DNA replication, RNA transcription, RNA modification and uncoating. They also include a few structural proteins.

Late phase

DNA synthesis

DNA synthesis occurs in "factories" and uses an unusual mechanism which will not be dealt with here.

Late transcription and translation

This is a complex process. Some late proteins are made throughout the late phase, but others only at the beginning of late phase. Some early proteins are not synthesized once DNA replication commences while other early proteins are made during late as well as early phases. Thus, there is complex control of which proteins are made by vaccinia and when they are made. This means that there are controls other than just early/late controls. (This is a very large virus, thus the complexity is not surprising.)

Assembly

Assembly occurs in "factories" in the cytoplasm. The new, immature virus particles acquire a membrane while in the cytoplasm - the exact mechanism is not fully understood but it seems that the virus gets "wrapped" by cellular membranes (figure 22). The older idea that a membrane is formed directly from lipids rather than from a pre-existing membrane is not correct. There is a gradual maturation of enveloped particles. The virus is usually released by host cell disintegration, but some may get out by budding through membranes (in which case they have an extra membrane). Both forms appear to be infectious. The exact mechanism by which the virus gets out of infected cells may depend on host cell type.

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