Replication of SV40 virus the model polyomavirus

The polyomaviruses have genomes of approximately 5000 base pairs. Capsids are made up of three proteins, usually called VP1, VP2, and VP3. Polyomaviruses can cause tumors in animals and can transform the growth properties of primary cells in culture, especially the cells from animals different from the virus's natural host (see Chapter 10, Part III). Polyomaviruses also stay persistently associated with the host, often with little evidence of extensive pathology or disease. Although these viruses kill the cells in which they replicate, this process is slow.

Polyomavirus

PE

Control

region

PL

0 1

100 1

200 1

300 1

TT

1 T 1

ori

"21"

"72"

Enhancer regions

Sv40 Genome

Fig. 16.1 Polyomavirus and the genetic and transcript map of SV40 virus. (a) The 60 pentameric subunits of the capsid proteins are arranged in an unusual fashion so that the packaging of individual capsomers is not equivalent in all directions. The drawing is based on computer-enhanced analysis using the electron microscope and x-ray diffraction methods (see Chapter 5) published by Salunke et al (Cell 1986;46:895-904). The 5243-base pair dsDNA genome is condensed with host cell histones and packaged into the 45-nm-diameter icosahedral capsid. (b) The early and late promoters, origin of replication, and bidirectional cleavage/ polyadenylation signals are shown along with the introns and exons of the early and late transcripts. A high-resolution schematic of the approximately 500-base pair control region with the early and late promoters is also provided. Two early promoter enhancers, one containing the 21-base pair repeats and the other containing the 72-base pair repeats, are shown. The origin of replication (ori) is situated between the enhancers and the early promoter, and the three binding sites for large T antigen (T) are indicated. (c) A higherresolution schematic of the processing of early viral mRNAs. Splice sites, translational reading frames, and other features are indicated by sequence number. Details are described in the text. Note that the 3' end of the pre-mRNA occurs just beyond the early polyadenylation site (2590) that is situated in the 3' transcribed region of the late pre-mRNA. (d) A higher-resolution schematic of the processing of late viral mRNAs. Splice sites, translational reading frames, and other features are indicated by sequence number. Details are described in the text. Note that the 3' end of the pre-mRNA occurs just beyond the late polyadenylation site (2650) and is situated in the 3' transcribed region of the early pre-mRNA. T-Ag=large T antigen; t-Ag=small t antigen.

In keeping with the requirement for extensive cellular function during replication, there is no global virus-induced shutoff of host function.

One widely studied polyomavirus is murine polyomavirus (Py), originally isolated from wild mice and named for its ability to cause many types of small tumors in some strains of newborn mice. Another widely studied polyomavirus is SV40 virus, which was originally named simian vacuolating agent 40. There is no evidence that this virus causes tumors in its natural host, but it can readily cause growth transformation of cells in culture. SV40 virus originally was found as a contaminant of African Green monkey kidney cells (AGMK) in which poliovirus was being grown for vaccine purposes. Early recipients of the Salk polio vaccine got a good dose of the virus, but no pathology has been ascribed to this, at least to the present time.

(c) Early SV40 2590 2693 pA

4572 4641 4917 5163

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1 IIIII^H

Overlaps 3'-end of late mRNA

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(Alternate splicing)

(Alternate splicing)

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(d) Late SV40 Ori

335 523 562 916

1499 1618

Agno

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Very late Upstream Agno protein transcript start

Agno and VP1 mRNA

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Agno and VP1 mRNA

Overlaps 3' end of early mRNA

Fig. 16.1 Continued

Whereas Rous sarcoma virus (a retrovirus) had been known to cause tumors in chickens since the early part of this century, the fact that its genome is RNA made understanding of its mechanism of oncogenesis out of the reach of molecular biologists working in the 1950s and 1960s. Indeed, major progress awaited the discovery of reverse transcriptase by Howard Temin and David Baltimore in 1970. By contrast, the fact that the DNA-containing SV40 and mouse polyomaviruses cause growth transformation and tumors in the laboratory provided a model for the study of the process that could be exploited with the techniques available at the time. The study of these viruses essentially launched the molecular biological study of carcinogenesis and eventually led to the discovery of tumor suppressor genes and their important role in regulating cell growth and division.

Its importance in fundamental research in oncogenesis, ease of manipulation in the laboratory, and convenient genome size have contributed to SV40 virus's status as, arguably, the most extensively studied of all DNA viruses. While Py and SV40 replication differ in some important features, the overall strategy is the same. Two human polyomaviruses, BK and JC, are known, and a third is suspected to exist but has not been rigorously identified. The BK and JC viruses are closely related to SV40, and are thought to be spread by the respiratory route. Primary infection occurs in children with little obvious pathology. In the United States, most children are infected with BK virus by the age of 5—6 years, and the only signs of infection may be a mild respiratory illness. Infection with JC virus occurs somewhat later, with most children being infected between the ages of 10 and 14 years.

Resolution of infection is complete in children with normally functioning cell-mediated immunity. Despite resolution, the virus persists for the life of the individual — one primary site of persistence is the kidney from which BK virus can be periodically shed. In addition, JC virus can be recovered from brain biopsy specimens. While this persistence has no known clinical manifestations in the healthy individual and is thought to be the result of viral genomes persisting in an inactive state in nondividing, terminally differentiated cells, immunosuppression by HIV infection or prior to organ transplantation can lead to severe consequences. In immune-compromised individuals, JC virus is associated with a rare progressive destruction of neural tissue in the CNS [progressive multifocal leukoencephalopathy (PML)]. This neuropathology is the result of the fact that transcription of the JC virus RNAs can take place in oligodendrocytes (but not other cells) in the adult brain, but just what aspect of immune suppression such as that engendered by HIV infection reactivates this dormant virus is unknown. While not as firmly established, it is pretty certain that BK virus infections can lead to urinary tract pathologies in immune-compromised individuals.

The exact sequence of human JC virus isolated from individuals in various parts of the world varies enough to allow its use as a genetic population marker. Extensive studies on natural isolates show that individual variants are strongly associated with individual ethnic and racial population groups, and their movements throughout the world can be traced by the occurrence of specific virus variants. This means that the virus has been associated with the human population for an extremely long time, and that variants have arisen as populations have diverged.

The pattern of infection of young animals followed by virus persistence and shedding is quite characteristic of the infection of laboratory strains of mice with murine polyomavirus. One notable difference between the pathology of this virus and that of SV40, JC, and BK viruses, however, is that infections of suckling mice can lead to the formation of tumors, hence, the name polyomavirus. Genetic studies suggest that a major factor in the ability of the murine virus to cause tumors is the presence of specific endogenous retroviruses in the laboratory mouse strains, and while there is some suggestive evidence that human polyomaviruses can be associated with tumors, definitive evidence of causation is lacking.

The SV40 genome and genetic map

The SV40 virus genome contains 5243 base pairs, and its map showing essential features is displayed in Fig. 16.1(b). The genome is organized into four functional regions, each of which is discussed separately.

The control region This region covers about 500 bases and consists of the origin of replication, the early promo ter/enhancer, and the late promoter. The sequence elements in this region overlap to a considerable extent, but the bases specifically involved with each function can be located precisely on the genome. This has been done by making defined mutations in the sequence and analyzing their effects on viral genome replication and on expression of early and late genes.

The early promoter region contains a TATA box and enhancer regions (noted by 72-base and 21-base repeats). Surprisingly, the late promoter does not have a TATA box, and late mRNA initiates at a number of places within a 60- to 80-base region. The multiple start sites for late mRNA expressed from this "TATA-less" promoter provided one of the early clues that the TATA box functions to assemble transcription complexes at a specific location in relation to mRNA initiation. It is not clear exactly what substitutes for the TATA box in the late promoter, but it is thought that transcription complexes can form relatively readily throughout the region.

The origin of replication (ori) is about 150 base pairs in extent and contains several elements with a sequence critically linked by "spacers" whose length but not specific sequence is important in function of the origin. The ori elements have some dyad symmetry; that is, sequence of the far left region is repeated in the inverse sense in the far right region. This symmetry is thought to have a role in allowing the DNA helix to "melt" at the origin, facilitating the entry of replication enzymes to begin rounds of DNA replication. The general process was described in Chapter 13, Part III.

The early transcription unit The SV40 genome's early region is shown in high resolution in Fig. 16.1(c). It is transcribed into a single mRNA precursor that extends about halfway around the genome, and contains two open translational reading frames (ORFs). The single early pre-mRNA transcript can be spliced at one of two specific sites (i.e., the pre-mRNA is subject to alternative splicing — see Chapter 13, especially Fig. 13.7). If a short intron is removed, an mRNA is generated that encodes a relatively small (approximately 20,000 daltons) protein (t (small t) antigen), which has a role in allowing the virus to replicate in certain cells.

A slightly smaller (and more abundant) mRNA is generated by the splicing of a larger intron in the pre-mRNA. This removes a translation terminator that terminates the small-t-antigen ORF. The smaller (!) mRNA encodes the T (large T) antigen (approximately 80,000 daltons). The large T antigen has a number of functions, including the following:

1 Activation of cellular DNA and RNA synthesis by binding to the cellular growth control gene products named Rb and p53. This binding stops these control proteins from keeping the cell contact inhibited. This function causes the infected cell to begin a round of DNA replication.

2 Blockage of apoptosis that is normally induced in cells where p53 is inactivated at inappropriate times in the cell cycle.

3 Binding to the SV40 ori to initiate viral DNA replication.

4 Shutting off early viral transcription by binding to regions in and near the early promoter.

5 Activating late transcription.

6 Playing a role in virion assembly.

The late transcription unit Late mRNA is expressed from a region extending around the other half of the genome from the late promoter; this is shown in Fig. 16.1(d). The late region contains two large ORFs that encode the three capsid proteins. Part of the expression of late proteins, then, requires alternate splicing patterns, just as is seen with the generation of early mRNA. Splicing of a large intron from the primary late pre-mRNA transcript generates an mRNA that encodes the 36,000-dalton major capsid protein (VP1). A small amount of mRNA is generated by splicing a small intron near the 5' end of the mRNA, allowing the first ORF to be translated into the 35,000-dalton VP2 protein.

The third capsid protein, VP3, is also expressed from the same mRNA encoding VP2 by utilization of an alternative translation initiation site. Ribosomes sometimes "miss" the first AUG of the 5' ORF in the mRNA expressing VP2. When this happens, the ribosome initiates translation at an AUG in phase with the first one but downstream, producing the 23,000-dalton VP3 protein. Thus, one mRNA encodes both VP2 and VP3, depending on where the ribosome starts translation. This "skipping" does not violate the general rule that a eukaryotic ribosome can only initiate a protein at the 5' ORF, as the first AUG is not seen and thus is in the operational leader sequence of the mRNA.

There is a fourth late protein expressed from the late region, but this is only seen very late in infection. This basic protein, the "agnoprotein," is encoded in a short ORF upstream of that encoding VP2. Very late in infection, some mRNAs are produced by initiation of transcription farther upstream than at earlier times, and these can be translated into this protein. The role of this product is not fully understood, but it may be involved in allowing the virus to replicate in certain cells that are normally nonpermissive for viral replication.

The polyadenylation region About 180 degrees around the circular SV40 genome from the ori/promoter region lies a second cis-acting control region. It contains polyadenylation signals on both DNA strands so that transcripts transcribed from both the early and late regions terminate in this region. It is notable that the polyadenylation signals for the mRNAs are situated such that the early and late transcripts have a region of 3' overlap. This can lead to the generation of dsRNA during the replication cycle, with attendant induction of interferon in infected cells (see Chapter 8).

Productive infection by SV40

Productive infection by SV40 in its normal host can be easily studied in cell culture using monkey kidney cells. The replication cycle is quite long, often taking 72 hours or more before cell lysis and release of new virus occur. One reason for this "leisurely" pace is that the virus is quite dependent on continued cellular function during most of its replication. The virus replicates efficiently in cultured cells that are actively dividing either because they have not yet reached confluence or because the cells are growth transformed and not subject to contact inhibition of growth. (The basic growth properties of cultured cells are discussed in Chapter 10, Part III.)

While the virus replicates efficiently in replicating cells, it also is able to replicate well in cells that are under growth arrest. This is by virtue of T-antigen expression early in infection. Manifestations of this ability provide many useful insights into the nature of the cell's ability to control and regulate its own DNA replication, and led to the discovery of the tumor suppressor genes p53 and Rb discussed in a following section.

Virus attachment and entry The replication cycle of SV40 is outlined in Fig. 16.2. Virions interact with a specific cellular receptor. This leads to receptor-mediated endocytosis, and the partially uncoated virion is transported in the endocytotic vesicle to the nucleus where viral DNA is released.

LATE

Lysis and release

LATE

Lysis and release

Sv40 Genome Map

Agno protein

Fig. 16.2 The replication cycle of SV40 virus in a permissive cell. The replication is divided into two phases, early and late. During the early stages of infection, virus attaches and viral genomes with accompanying cellular histones are transported to the nucleus via receptor-mediated endocytosis. RNA polymerase II (pol II) recognizes the enhanced early promoter, leading to transcription of early pre-mRNA, which is processed into mRNAs encoding small t (t-Ag) and large T antigen (T-Ag). These mRNAs are translated into their corresponding proteins. Large T antigen migrates to the nucleus where it carries out a number of functions, including inactivation of the cellular growth control proteins p53 and Rb, and binding of the SV40 origin of DNA replication (ori). Viral DNA replication takes place by the action of cellular DNA replication enzymes, and each round of DNA replication requires large T antigen to bind to the ori.

As genomes are replicated, the late stage of infection begins. High levels of large T antigen suppress the expression of early pre-mRNA and stimulate expression of late pre-mRNA. This is processed into two late mRNAs; the smaller encodes both VP2 and VP3 while the larger encodes VP1. At very late times, some transcripts are expressed and can be translated into the small agnoprotein. Viral capsid proteins migrate to the nucleus where they assemble into capsids with newly synthesized viral DNA. Finally, progeny virus is released by cell lysis.

Agno protein

Fig. 16.2 The replication cycle of SV40 virus in a permissive cell. The replication is divided into two phases, early and late. During the early stages of infection, virus attaches and viral genomes with accompanying cellular histones are transported to the nucleus via receptor-mediated endocytosis. RNA polymerase II (pol II) recognizes the enhanced early promoter, leading to transcription of early pre-mRNA, which is processed into mRNAs encoding small t (t-Ag) and large T antigen (T-Ag). These mRNAs are translated into their corresponding proteins. Large T antigen migrates to the nucleus where it carries out a number of functions, including inactivation of the cellular growth control proteins p53 and Rb, and binding of the SV40 origin of DNA replication (ori). Viral DNA replication takes place by the action of cellular DNA replication enzymes, and each round of DNA replication requires large T antigen to bind to the ori.

As genomes are replicated, the late stage of infection begins. High levels of large T antigen suppress the expression of early pre-mRNA and stimulate expression of late pre-mRNA. This is processed into two late mRNAs; the smaller encodes both VP2 and VP3 while the larger encodes VP1. At very late times, some transcripts are expressed and can be translated into the small agnoprotein. Viral capsid proteins migrate to the nucleus where they assemble into capsids with newly synthesized viral DNA. Finally, progeny virus is released by cell lysis.

The association of viral genomic DNA with cellular chromosomal proteins is a common feature in the replication of nuclear replicating viruses discussed in this and the next chapter. In the case of SV40 and other papovaviruses, the viral DNA is associated with histones and other chromosomal proteins when it is packaged into the virion. It remains associated with chromosomal proteins upon its entry into the nucleus. This means, in effect, that viral DNA is actually presented to the cell as a small or "mini"-chromosome. Essentially then, the cell's transcriptional machinery recognizes the viral chromosome and promoters therein merely as cellular genes waiting for transcription.

Early gene expression Early gene expression results in formation of large quantities of large-T-antigen mRNA, and less amounts of small-t-antigen mRNA. The amounts of protein synthesized are roughly proportional to the amount of mRNA present. The small t antigen contains the same N-terminal amino acids as does large T antigen because of the way early pre-mRNA is spliced into the two early mRNAs, as shown in Fig. 16.1(c). The splice-generating mRNA that encodes the T antigen removes a translation stop signal. By contrast, the splice in the tantigen mRNA is beyond the ORF, and thus does not affect protein termination. Generation of two proteins with major or minor differences in function but with a shared portion of amino acid sequence is quite common with many viruses. It is very important in the expression of adenovirus proteins.

The role of Tantigen in viral DNA replication and the early/late transcription switch As outlined in the preceding section, T antigen alters the host cell to allow it to replicate viral DNA. The T antigen also binds to the SV40 ori to allow DNA replication to begin, and to shut off synthesis of early mRNA. Each round of DNA replication requires T antigen to bind to the origin of DNA replication and initiate a round of DNA synthesis. DNA replication then proceeds via leading and lagging strand synthesis using cellular enzymes and proteins as described in Chapter 13. Since the SV40 genome is circular, there is no end problem, and the two daughter circles are separated by DNA cleavage and ligation at the end of each round of replication. This resolution of the interlinked supercoiled DNA molecules into individual genomes is mediated by cellular enzymes, notably topoisomerases and resolvases. The process is illustrated in Fig. 16.3. It is important to note that association of the daughter DNA genomes with cellular histones is not shown in the figure, but this association is necessary for the virus to be efficiently encapsidated.

While DNA replication proceeds, the relative rate of early mRNA synthesis declines owing to accumulation of increasing amounts of large T antigen in the cell, which represses synthesis of its own mRNA by binding at the ori and early promoter. While the relative amount of early mRNA declines in the cell at late times, its production never entirely ceases because there is always some template that has not yet bound large T antigen available for early mRNA expression.

At the same time that this versatile protein is modulating and suppressing its own synthesis, it activates transcription of late pre-mRNA from replicating DNA templates. Late transcripts have heterogeneous 5' ends, and as noted previously, very late in infection, the start of late mRNA transcription shifts to a point upstream of that previously used and the agnogene protein (the agnoprotein) can be encoded and translated from a novel subset of late mRNAs.

Abortive infection of cells nonpermissive for SV40 replication

Relatively early in the study of polyomavirus replication, infection of cells derived from a species other than the natural host of SV40 was observed to lead to an abortive infection where no virus was produced. Despite this, virus infection was shown to stimulate cellular DNA

  1. 16.3 The replication of SV40 DNA. The closed circular DNA has no end problem, unlike the replication of linear DNAs. Structures of the replication fork and growing points are essentially identical to those in replicating cellular DNA, and use cellular DNA replication enzymes and accessory proteins. Replication results in the formation of two covalently closed and interlinked daughter genomes that are nicked and religated into individual viral genomes by the action of cellular topoisomerase and other helix-modifying enzymes. T-Ag=large T antigen; ori = origin of replication.
  2. 16.3 The replication of SV40 DNA. The closed circular DNA has no end problem, unlike the replication of linear DNAs. Structures of the replication fork and growing points are essentially identical to those in replicating cellular DNA, and use cellular DNA replication enzymes and accessory proteins. Replication results in the formation of two covalently closed and interlinked daughter genomes that are nicked and religated into individual viral genomes by the action of cellular topoisomerase and other helix-modifying enzymes. T-Ag=large T antigen; ori = origin of replication.

replication and cell division, and study of this phenomenon provided early important models for the study of carcinogenesis. While such abortive infections may be purely a laboratory phenomenon, the information derived from them provided an important foundation for understanding the pathogenesis of papovaviruses in their natural hosts and viral oncogenesis.

In rodent (and some other nonprimate) cells, SV40 virus can infect and stimulate cellular RNA and DNA synthesis by expressing the large T antigen. As noted, this viral protein inactivates at least two cellular tumor suppressor or growth control genes (p53 and Rb). The role of such oncogenes in controlling cell growth is briefly touched on in Chapter 10, Part III, and is discussed in more detail in Chapter 19.

The two proteins in question (p53 and Rb) have two basic functions. First, they mediate an active repression of cell division by binding to and thus inactivating cellular proteins required to initiate such division. Second, levels of the free proteins above a critical level induce apoptosis (programmed cell death, see Chapter 10) in the cells that escape repression and begin to divide. As in the early phase of productive infection, in the first stages of infection of the nonpermissive cells, large T antigen displaces active replication-initiation proteins bound to p53 by binding this protein with higher avidity. The proteins thus liberated are free to initiate cellular DNA replication, but since there is no free p53, there is no induction of apoptosis.

These are the same steps that occur in the early stages of productive infection; however, viral DNA cannot be replicated in the nonpermissive cells. This failure is due to the inability of T antigen to interact effectively with one or more of its other cellular targets important in the early phases of infection. In this abortive infection, the cells in which T antigen is expressed do not die, but they replicate even while in contact with neighboring cells; this process is shown in Fig. 16.4. The continued stimulation of cellular DNA replication by expression of viral T antigen can lead to continual cell replication (i.e., transformation). Stable transformation will require the viral genome to become stably associated with cellular DNA by integration of viral DNA into the cellular genome. Such viral DNA replicates every time the cell replicates, and thus keeps the cell transformed.

The integration of viral DNA into a host cell chromosome is not a function of T antigen or any other viral product. Indeed, most abortively infected cells will divide for a round or so until the viral DNA is lost, and then they will revert to their normal growth characteristics. This is sometimes termed transitory (transient or abortive) transformation. The integration of viral DNA into the host cell is the result of an entirely random recombination event and occurs at sites where a few bases of the circular viral DNA can anneal to a few bases of chromosomal DNA. This must be followed by breakage and religation of the chromosome with the incorporated viral DNA. Obviously, this does not occur very frequently, but if a large number of cells are abortively infected with the polyomavirus in question and one or more integrate the viral chromosome and continue to express T antigen, those cells will form a focus of transformation. Such a focus is a clump of transformed cells growing on the surface of a culture dish of contact inhibited cells. These foci can be counted and are subject to similar statistical analyses as are plaques formed by productive infection. Some typical foci of transformation are shown in Fig. 10.6.

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  • letteria
    Where does jc polyomavirus replicate?
    1 year ago

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