MicrobiologyBytes: Virology: Polyomaviruses Updated: April 8, 2009 Search

Polyomaviruses

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Polyomaviruses infect a wide variety of vertebrates (12 members now known). Murine polyomavirus was isolated by Ludwig Gross in 1953 while he was studying leukaemia in mice and named because it caused solid tumours at multiple sites. The second member of the family, Simian Vacuolating Virus 40 (SV40) was isolated by Sweet and Hilleman in 1960 in primary monkey kidney cells cultures being used to grow Sabin OPV (Hilleman MR. Discovery of simian virus 40 (SV40) and its relationship to poliomyelitis virus vaccines.Dev Biol Stand 94: 183-190, 1998). Two human polyomaviruses were isolated in 1971:

  • BK Virus (BKV) - from an immunosuppressed kidney transplant patient
  • JC Virus (JCV) - from a case of progressive multifocal leukoencephalopathy (PML)
  • New human polyomaviruses were discovered in 2007

Because of the small size of their genomes, their oncogenic properties and the existence of in vitro systems, polyomaviruses have been used extensively as models for DNA replication.

Morphology:

Polyomaviruses virions are non-enveloped, ~45nm diameter T=7 icosahedral particles. The complete structure of SV40 has been described. There are 3 capsid proteins, VP1-3, which form 72 pentameric capsomers, 60 hexagonally co-ordinated plus 12 pentamerically co-ordinated (at the vertices):

Computer reconstruction of papovavirus particle

There are 3 capsid proteins, VP1-3, which form 72 pentameric capsomers, 60 hexagonally co-ordinated + 12 pentamerically co-ordinated (at the vertices).

Each virion contains 360 copies of VP1 (i.e. 72 x 5) + 30-60 copies each of VP2 & VP3, i.e. ~1 copy/pentamer. Each copy of VP1 has a sialic acid binding site on the surface & these form the receptor-binding site for the virus; hence the particles have haemagglutinating properties.

VP2/3 have overlapping sequences (see below) - VP2 contains the entire sequence of VP3 at its C-terminus, +115 aa at the N-terminus. The precise location of VP2/3 is unknown. VP2 is myristylated at its NH2-terminus, which is believed to be important in holding the particle together (c.f. picornavirus VP4).

Genome:

Polyomavirus genomes are d/s, circular DNA molecules, ~5kbp in size. The entire nucleotide sequence of all the viruses in the family is known and the architecture of the Polyomavirus genome (i.e. number and arrangement of genes and regulatory signals and systems) has been studied in detail.

Within the particles, the DNA assumes a supercoiled form (like plasmid DNA). Four cellular histones H2A, H2B, H3 and H4 are associated with the DNA.

Polyomavirus genomic organization is designed to pack maximal information (6 genes) into minimal space (5kbp). This paradox is achieved by the use of both strands of the genome DNA and overlapping genes:

SV40 genome

The origin of replication is surrounded by non-coding regions which control transcription. VP1 is a encoded by a "dedicated" ORF, but the VP2 and 3 genes overlap so that VP3 is contained within VP2. SV40, BKV and JCV encode a small (60-70aa) protein known as the agnoprotein which enhances assembly of virus particles & cell to cell spread.
Polyomaviruses also encode "T-antigens" - proteins which can be detected by sera from animals bearing polyomavirus-induced tumours:

Protein Size (a.a.):
Virus: Large T: Small T: Middle T:
Polyoma 785 195 421
SV40 708 174 ---
BKV 695 172 ---
JCV 688 172 ---

These proteins have common N-terminal regions but unique COOH-termini derived from alternative splicing patterns.

Replication:

PyV genomes are d/s, circular DNA molecules, ~5kbp in size. The entire nucleotide sequence of all the viruses in the family is known & the architecture of the PyV genome (i.e. number & arrangement of genes & regulatory signals & systems) has been studied in detail.

Within the particles, the DNA assumes a supercoiled form (like plasmid DNA). Four cellular histones H2A, H2B, H3 & H4 are associated with the DNA.

The genome is functionally divided into 3 regions:

  • Early: Expressed early in virus infection, i.e. BEFORE genome replication. Expression of early genes continues during the late stage of infection. Encodes non-structural proteins (i.e. not present in virus particle).
  • Late: Expressed later in virus infection, i.e. DURING & AFTER genome replication. Encodes structural proteins (i.e. present in virus particle).
  • Regulatory region: Contains transcriptional promoters & enhancers plus the unique origin of DNA replication.
  • TEMPORAL CONTROL (i.e. EARLY vs. LATE) OF GENE EXPRESSION IS A COMMON FEATURE OF CLASS I VIRUSES.

    Polyomavirus replication

    Attachment:

    SV40 receptor appears to be MHC class I antigen(s).
    Receptors for PyV are not known, but appear to contain sialic acid (haemagglutination) & be widespread in many tissue/species.
    VP1 (only(?) external protein on virus capsid) responsible for receptor binding (anti-VP1 Abs block binding).

    Entry:

    VP2/3 are myristylated & believed to interact with cellular membranes to facilitate entry.
    Virions are taken up by endocytosis & is transported to the nucleus by interaction of endocytic vacuoles with the cytoskeleton.

    Uncoating:

    Virus particles enter by the nuclear pores & uncoating occurs inside the nucleus.
    The rest of the replication cycle occurs in the nucleus. VP2/3 mutants defective in uncoating, therefore these proteins are involved in the process, although the details are unknown.
    How Do Animal DNA Viruses Get To The Nucleus? Ann.Rev.Microbiol. (1998) 52: 627-686

    Gene Expression:

    Inside the nucleus, the virus mini-chromosome (genome-histone complex) is transcribed by host cell RNA polymerase II to produce early mRNAs.
    Because of the relative simplicity of the genome, PyV are heavily dependent on the cell for transcription & genome replication. However, the genome contains cis-acting regulatory signals (surrounding the origin of replication) which direct transcription, & trans-regulatory proteins (the T-antigens) which direct transcription & replication. Alternative splicing produces 2 (or 3) species of early mRNA/T-antigen:
    large-T & small-T (plus middle-T in murine polyomavirus - a membrane protein found in the plasma membrane, important in cell transformation).
    Transcription from the early region promoter is autoregulated by binding of large-T antigen to the regulatory region of the genome:

    Polyomavirus expression

    The early gene promoter contains strong enhancer elements which cause it to be active in newly infected cells. The early region proteins are the T-antigens.
    Small T-antigen is not essential for virus replication, but indirectly (i.e. interacts with cellular proteins but does not bind directly to virus genome) enhances transcription from the late promoter.
    As the concentration of large T-antigen builds up in the nucleus, transcription of the early genes is repressed by direct binding of the protein to the origin region of the virus genome, preventing transcription from the early promoter and causing the switch to the late phase of infection. After DNA replication has occurred, transcription of the late genes occurs from the late promoter and results in the synthesis of the structural proteins, VP1, VP2 and VP3.
    The SV40 late promoter is a very strong promoter & is activated by binding of large T-antigen to the 72bp repeats upstream of the transcription start site.
    Therefore, the role of the SV40 T-antigen in controlling the transcription of the genome is comparable to that of a 'switch'.

    Genome Replication:

    Large T-antigen has a complex action & binds to various cellular proteins:

    SV40 DNA replication is initiated by binding of large T-antigen to the origin region of the genome. The function of T-antigen is controlled by phosphorylation, which decreases the ability of the protein to bind to the SV40 origin.
    The SV40 genome is very small and does not encode all the information necessary for DNA replication. Therefore, it is essential for the host cell to enter S phase, when cell DNA and the virus genome are replicated together.
    Protein:protein interactions between T-antigen and DNA polymerase-alpha directly stimulate replication of the virus genome. Inactivation of tumour suppressor proteins bound to T-antigen causes G1-arrested cells to enter S phase, promoting DNA replication.
    Therefore, in addition to increasing transcription, another function of T-antigen is to alter the cellular environment to permit virus genome replication.

    Assembly/Maturation:

    Virus proteins contain 'nuclear localization signals' which results in their accumulation in the nucleus, where they migrate after being synthesized in the cytoplasm.
    Assembly occurs in the nucleus. Since the structure of the virus is relatively simple, assembly & maturation of the particle are simultaneous.

    Release:

    Some virus particles are exported to the cell surface in cytoplasmic vacuoles. The remaining virus is released when the cell lyses (SV40!). Mechanism of cell injury is not clear, but is not a surprise due to the severe interference with normal cellular metabolism & growth that these viruses cause.
    A Very Late Viral Protein Triggers the Lytic Release of SV40. 2007 PLoS Pathogens 3, 7, e98
    The complete replication cycle takes 48-72h (depending on multiplicity of infection).

     

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    Pathogenesis:

    Infection of cells by PyV can result in two outcomes: The outcome appears to be determined primarily by the cell type infected.
    However, after infection, some (unknown) determinant in the intracellular environment (rather than the receptor-V.A.P. interaction) determines the outcome of the infection:

    Two out of six proteins encoded by SV40, termed large T and small t antigens, are involved in immortalisation and transformation of primary rodent cells. Although SV40 large T antigen is sufficient to cause immortalisation and transformation of primary rodent cells, small t antigen enhances large T-induced transformation. SV40 large T and small t antigens are products of the same gene generated by alternative splicing and they share an 82 amino acid N-terminus. The SV40 large T antigen is a 708 amino acid nuclear phosphoprotein with multiple biochemical activities. It is composed of several domains, three of which contribute to transformation of cells in culture or formation of tumours in animals. These three domains have been localised to amino acids 1-82 (Region 1), 101-118 (Region 2) and amino acids 351-450 and 533-626 (Region 3):

    Small t antigen of SV40 comprises 174 amino acids. The region between residues 97-103 interacts with the protein phosphatase 2A (PP2A). This interaction reduces the ability of PP2A to inactivate ERK1 and MEK1 protein kinases, resulting in stimulation of proliferation of quiescent monkey kidney cells. Small t antigen-dependent assays also identified other regions which had the ability to enhance cellular transformation. These regions are located in the N-terminal part which is shared by the small and large T antigens of SV40 and can potentially function as a Dna J domain. Small t antigen can also associate with tubulin and it has been suggested that this plays a role in its biological function.

    The polyomaviruses encode three proteins involved in cellular transformation termed:

    These three proteins result from the differential splicing of the early region transcript and contain homologous sequences. The large T antigen of polyoma interacts with the tumour suppressor protein, pRb and is able to immortalise primary fibroblasts in culture. The Dna J domain located at its N-terminus, particularly the HPDKYG sequence found between residues 42 and 47, is critical for functional inactivation of Rb family proteins, as is also the case with SV40 large T antigen. The expression of LT is not sufficient to produce a fully transformed cell phenotype - this requires mT, which is the major transforming protein of the polyomavirus. Mouse polyoma middle T consists of 421 amino acids and can be divided into at least three domains, some of which are shared with LT and sT. The amino terminal domain is comprised of the first 79 amino acids and is also present in LT and sT. Adjacent to it, between residues 80-192, is a domain that is also present in the polyoma sT and contains two cysteine rich regions, Cys-X-Cys-X-X-Cys, which have also been identified in small t of SV40. Mutation of these cysteines abolishes the ability of mT to transform cells. The remaining 229 amino acids are unique to mT and contain the major tyrosine phosphorylation site of mouse mT and a hydrophobic region (approximately 20 amino acids at the carboxy-terminus) involved in membrane localisation of this protein which is necessary for its transforming activity.

    Polyoma mT associates with several cellular proteins. These complexes include protein phosphatase 2A (PP2A), Src family tyrosine kinases, phosphatidylinositol 3-kinase (PI-3K) and an adaptor protein, Shc, and they are necessary for cellular transformation.The existence of large complexes containing mT and all the cellular targets described above has been reported. Most of the mT in a transformed cell is complexed with the serine/threonine-specific protein phosphate PP2A, which also associates with the small T antigens of polyoma and SV40. PP2A usually consists of the core dimer of a 36 kDa catalytic subunit (C subunit) and a regulatory subunit of 65 kDa (A subunit) which associate with variable regulatory subunits (B subunit) to give a heterotrimer. Polyoma mT and both polyoma and SV40 small T antigens displace the variable B subunit in this enzyme, although there is no sequence homology between them and this subunit. The interaction of PP2A with these tumour antigens has an inhibitory effect on dephosphorylation of in vitro substrates such as myosin light chain, myelin basic protein, large T antigen and p53 and also reduces its ability to inactivate ERK1 and MEK1 protein kinases, which results in cell cycle progression.

    About 10% of mT in a transformed cell is bound to pp60c-src (c-Src). This cytoplasmic protein kinase has several non-catalytic domains which regulate its enzymatic activity in both a positive and a negative fashion. In normal cells, the activity of c-Src is repressed by phosphorylation of Tyr527 in the non-catalytic C-terminal region which has been mapped as the mT binding site. Molecules of c-Src complexed with mT are not phosphorylated at Tyr527 and this is accompanied by a ten-fold increase in kinase activity. Genetic analysis has shown that association of mT and c-Src is necessary but not sufficient for transformation.

    Phosphatidylinositol 3-kinase (PI 3-K) is another enzyme with which mT can form a complex. PI 3-K consists of two subunits: a catalytic subunit of 110 kDa (p110) and a regulatory subunit of 85 kDa (p85). The regulatory subunit, p85, contains two SH2 (Src Homology) and one SH3 domain. Binding of mT to PI 3-K occurs through the interaction of the SH2 domains of p85 with the PTyr315XXMet sequence of mT when Tyr315 is phosphorylated by the associated Src family kinases. It has been recently reported that mT activates the Ser/Thr kinase Akt in a PI3-kinase-dependent manner

    The mouse polyoma middle T also interacts with a family of adaptor proteins (66, 52 and 46 kDa) termed Shc for Src Homology 2 (SH2) and Collagen (a1) related. A single mutation in middle T antigen which replaces tyrosine at position 250 by serine disrupts the association of this protein with Shc and results in much reduced transformation of cells in culture and alters the spectrum of tumours and their morphology in inoculated animals. Phosphorylated Shc associates with another adaptor protein, Grb2, which is able to bind to SOS, the 150 kDa guanylnucleotide exchange factor for Ras. This targets SOS to the plasma membrane location of Ras allowing the rapid conversion of Ras from the inactive GDP-bound to the active GTP-bound state. Transformation by the mouse middle T requires functional Ras. However the hamster middle T does not have a region homologous to the segment containing tyrosine 250 of mouse middle T, suggesting that the Shc link may not be involved in its transforming mechanism.

    Polyoma middle T antigen can also form complexes with phospholipase C-g-1 and 14-3-3 proteins. Tyrosine 322 of mT, when phosphorylated, becomes the docking site for the SH2 domain of phospholipase C-gamma-1 (PLC-g-1). Mutation of tyrosine 322 to phenylalanine renders mT defective for interaction with PLC-g-1 and in transformation (18). Phosphorylation of serine 257 of mT regulates its association with 14 -3-3 proteins, which have been linked to cell cycle control and signalling.

    Transformation is a rare and accidental consequence of the sequestration of tumour suppressor proteins. Inactivation of tumour suppressor proteins bound to T-antigen causes G1-arrested cells to enter S phase and divide and this is the mechanism which results in transformation. However, the frequency with which abortively infected cells are transformed is low (about 1x10-5).

    Therefore, T-antigen:

  • Alters the cellular environment, affecting the cell cycle & DNA replication, enhancing virus replication (simple genomes!)
  • Accidentally may result in cellular transformation
  • Viral DNA replication is initiated by binding of large T-antigen to the origin, replication then proceeds bidirectionally from this point - IMPORTANT MODEL FOR CELLULAR DNA REPLICATION/ONCOGENESIS.

    MicrobiologyBytes: Is There a Role for SV40 in Human Cancer?
    Shah KV. SV40 and human cancer: a review of recent data. Int J Cancer. 2007 120: 215-223: An unknown proportion of formalin-inactivated poliovirus vaccine lots administered to millions of US residents between 1955 and 1963 was contaminated with small amounts of infectious simian virus 40 (SV40), a polyomavirus of the rhesus macaque. It has been reported that mesothelioma, brain tumors, osteosarcoma and non-Hodgkin lymphoma (NHL) contain SV40 DNA sequences and that SV40 infection introduced into humans by the vaccine probably contributed to the development of these cancers. In summary, the most recent evidence does not support the notion that SV40 contributed to the development of human cancers.

    Alternatively, two polyomaviruses commonly infect man and have been associated with disease.

    Human infections

    Site of primary infection is not known, but may be the respiratory tract. The implications of this are that the vast majority of primary infections with these viruses are asymptomatic. However, both viruses are oncogenic when inoculated into newborn hamsters. Once infected, the viruses persist (for life?) and disease appears to be associated with reactivation rather than primary infection. Pregnancy is known to reactivate Polyomaviruses infections, but without any known pathologic consequence.

    JC Virus

    Associated with progressive multifocal leukoencephalopathy (PML). This is a rare disease, involving plaques of demyelination/inflammation in the CNS. Oligodendrocytes from these lesions (responsible for the synthesis and maintenance of the myelin sheath around neurons) are productively infected with JCV. The disease is seen in two main groups of people:

    MedScape: Screening to Prevent Polyoma Virus Nephropathy

    BK Virus

    Primary infection is associated with a mild respiratory illness in children. The virus has also been isolated from various human tumours, but a cause and effect relationship has not been demonstrated. A recent report has suggested that up to 10% of kidney transplants fail as a result of BK virus infection (Hirsch HH, et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation. 79:1277-1286, 2005).

    New Human Polyomaviruses

    In 2007, a third polyomavirus, KI, was described in human clinical specimens, although its pathogenicity and prevalence in humans has not yet been established (Identification of a third human polyomavirus. 2007 J Virol 81: 4130–4136). Subsequently, another novel polyomavirus (WU) was found in respiratory secretions from human patients with symptoms of acute respiratory tract infection. Are KI and WU viruses human pathogens? If so, what kind of disease does they cause? Perhaps most importantly, there are likely to be many more as of yet unidentified viruses infecting the human body (Identification of a Novel Polyomavirus from Patients with Acute Respiratory Tract Infections. PLoS Pathogens 2007 3 (5): e64).



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