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INFECTIOUS DISEASE

BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

VIDEO LECTURE

VIROLOGY -  CHAPTER  THREE   

DNA VIRUS REPLICATION STRATEGIES  

Dr. Margaret Hunt 

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Logo image © Jeffrey Nelson, Rush University, Chicago, Illinois  and The MicrobeLibrary

 READING:
Murray et al., Microbiology
 5th Ed., Chapter 52-56

 

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.

 

 

 

NUCLEAR DNA VIRUSES  

PARVOVIRUS FAMILY

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

papilloma3.gif (15776 bytes)  Figure 1 Papilloma virus © Dr Linda Stannard, University of Cape Town, South Africa. Used with permission

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

 

Properties of POLYOMaviruses and Papillomaviruses

They are small: 40 - 60nm
They are icosahedral: major capsid protein is VP1, with lesser amounts of VP2, VP3
They are non-enveloped
They have circular, double-stranded DNA is associated with cell histones (nucleosomes)

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

 

 

dna2.jpg (329086 bytes) Figure 2 SV40 virus, a polyoma virus © Dr J-Y Sgro, University of Wisconsin. Used with permission

POLYOMAVIRUSES

These include SV40, 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.

 

dna3.jpg (155321 bytes) 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)

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)

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.

 

 

dna5.jpg (260900 bytes) Figure 5 VP1, 2, and 3 are made from same primary transcript which undergoes differential splicing. This results in the reading frame for VP1 being different from that for VP2 and VP3
dna6.jpg (214612 bytes)  Figure 6
Gene expression by SV40.
Early genes are in red, late genes are in green.  Note: - - - - indicates regions of the primary transcript which are removed in the alternatively processed mRNA. Cross-hatched area indicates region of RNA translated in different reading frames according to which alternatively spliced transcript is being translated Modified from Fiers et al.,Nature 273:113  
 

FEATURES TO NOTE ABOUT POLYOMA VIRUS STRATEGY

Early and late functions
Multiple use of the same DNA sequence (alternative splicing, overlapping reading frames)
Multifunctional protein (T antigen)
Small genome - so not surprising that virus codes for a very limited number of proteins
Host cell provides RNA synthesis machinery, RNA modification machinery, DNA synthesis machinery, histones for packaging DNA.

 

 

adeno2.gif (35105 bytes) Figure 7a Adenovirus structure © Dr Linda Stannard, University of Cape Town, South Africa. Used with permission

dna7.jpg (25995 bytes) Figure 7b Adenovirus structure

ADENOVIRUSES

 

PROPERTIES OF ADENOVIRUSES

Larger than papovaviruses (70nm diameter)
Non-enveloped, icosahedral viruses with fibers at vertices (figure 7 and 8)

Genome about 7 times size of polyoma virus genome

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

 

 

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
dna8.jpg (116082 bytes)  
B
adeno-diag.jpg (116419 bytes) 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

 
adeno-uncoat.jpg (166044 bytes) Figure 9  Diagrammatic representation of the uptake and uncoating of adenovirus particles. Adapted from Zinsser Microbiology 20th Ed. 
dna11.jpg (181977 bytes) 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

 

dna12.jpg (423570 bytes) Figure 11 Adenovirus DNA replication by a displacement mechanism

 

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.

 

dna13.jpg (203007 bytes) Figure 12
Transcription map of adenovirus. Early genes are shown in red. Green indicates late genes. Blue lines indicate DNA. Square brackets indicate the promotors. Missing regions indicate removal of introns. Adapted from Broker, T.R. In Processing of RNA. (Apirion, D ed) 181-212, 1984

dna14.jpg (138942 bytes) Figure 13 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 and 13):

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.

 

   
FEATURES TO NOTE ABOUT ADENOVIRUS STRATEGY
Adenoviruses are larger and more complex than papovaviruses.
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.

 

dna15.jpg (627020 bytes) Figure 14a  Herpes virus structure

herpes.gif (48070 bytes) Figure 14b Herpes simplex virus  © Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission). 

 

 

 

HERPESVIRUSES  

 

PROPERTIES OF HERPES VIRUSES

Larger virions (180 - 200nm) than adenoviruses
Larger genome (three to five times) than adenoviruses
Linear, double-stranded DNA
Enveloped icosahedral virus (this means that lipid solvents readily inactivate these viruses) (figure 14)
Figure 15a Herpes simplex virus adsorbing to the plasma membrane  © Dennis Kunkel Microscopy, Inc.  Used with permission

dna16.jpg (333091 bytes) Figure 15b 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 (results in partial uncoating) (figure 15). Such fusion with the plasma membrane has implications for both the virus and the host cell. Among these are:

i) 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.

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

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

 

herpeslay.jpg (111723 bytes) Figure 16  Expression of immediate early, early and late genes of herpesviruses

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

 

 

dna19.jpg (158061 bytes) Figure 18 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 (figure 18). 

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 18), 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 19A
Herpes simplex virus in cellular vacuoles and cytoplasm of peripheral blood lymphocyte
 © Dennis Kunkel Microscopy, Inc.  Used with permission

Figure 19B
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 19). 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 19C  (right)
Stages in the exocytosis of herpes virus from the nucleus, in which the virus core is assembled, to the plasma membrane
 
dna20.jpg (259518 bytes)  Figure 20 Negative stain and thin section of pox viruses  © F. Fenner

  

CYTOPLASMIC DNA VIRUSES  

 

smallpox.jpg (23267 bytes) Figure 21 Boy with smallpox   CDC/Cheryl Tryon ctt1@cdc.gov 

POXVIRUSES  

There are several reasons why poxviruses (figure 20) are of importance:

  • Certain poxviruses are of historic note,  such as smallpox (figure 21) 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 22), orf, monkey pox, cow pox). Note: chicken pox is caused by a herpes virus which  is not a member of the poxviridae
pox-moll-cont.jpg (35295 bytes) Figure 22  Transmission electron micrograph of poxvirus of molluscum contagiosum  CDC 

PROPERTIES OF POXVIRUSES

large virions
large genome, double-stranded DNA
size varies but they are as larger as or larger than herpesviruses
complex morphology
enveloped
 


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.)

 

poxexo2.jpg (186107 bytes) Figure 23 Possible scheme for the formation of infectious pox virions. The virus core becomes wrapped in cytoplasmic membrane and may escape when the host cell is lyzed. Some other membrane-bound virions may bud through other membranes, in which case they have two membranes. In either case, the virions are infectious. Adapted from  Baron, S. Ed. Medical Microbiology 4th Edition. 1996. 

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 23). 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.

FEATURES TO NOTE ABOUT POXVIRUSES

Cytoplasmic
Large genome
The virus does a lot of things for itself
The virus has unusual capabilities compared to other viruses

 

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