Polyomaviruses

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Morphology:

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:

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:

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:

TEMPORAL CONTROL (i.e. EARLY vs. LATE) OF GENE EXPRESSION IS A COMMON FEATURE OF CLASS I VIRUSES.

Attachment:

Entry:

Uncoating:

Gene 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:

• DNA polymerase alpha & s/s DNA binding protein, both involved in DNA replication
• p53 & p105

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:

Release:

Pathogenesis:

• Productive (lytic) infection
• Non-productive (abortive) infection

• Polyomavirus: Lytic infection of mouse cells, abortive infection of rat/hamster cells.
• SV40: Lytic infection of monkey cells, abortive infection of mouse cells.

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):

• Region 1 is homologous to the J domain of the Dna J family of molecular chaperones. This region plays an essential role in virion assembly, viral DNA replication, transcriptional control and oncogenic transformation. It has, therefore, been proposed that the J domain is involved in the rearrangement of multiprotein complexes. Further studies have suggested that the J domain is required for functional inactivation of Rb family proteins.
• Region 2 of T antigen, which has been shown to be important for host cell transformation, contains a consensus amino acid sequence, LXCXE, that is required for binding to the tumour suppressor protein, pRb, and two structurally related proteins, p107 and p130. Large T antigens with mutations in this region are defective for pRb/p107/p130 binding and for transformation of REF52 and NIH 3T3 cell lines. However, as described above, further results suggest that the J domain of SV40 large T antigen plays a role in the functional inactivation of Rb family proteins. A model has therefore been proposed where Rb family proteins bind to Region 2, but the J domain directs their inactivation by perturbing the phosphorylation of p107 and p130 and promoting the degradation of p130.
• The Region 3 of SV40 large T contains a p53 tumour suppressor binding domain which is located between amino acids 351-626. A spacer segment redundant for binding to p53 divides this region into two parts between amino acids 351-450 and 533-626. In one model it is proposed that amino acids 351-450 are important for direct contact with p53 and amino acids 533-626 are important for regulating this interaction.
• A novel functional domain involved in preventing apoptosis independently of inactivation of p53 has recently been mapped to SV40 large T. The carboxy-terminal region of large T also binds p300, pCBP and p400 transcriptional coactivators and this activity has been implicated in the transforming mechanism of this protein.

The polyomaviruses encode three proteins involved in cellular transformation termed:

• large tumour antigen (LT)
• middle T antigen (mT)
• small tumour antigen (sT)

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:

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.

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

• the elderly
• immunocompromised hosts (transplant and AIDS patients).

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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• Medscape: Polyomavirus in Solid Organ Transplantation
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