The Hamster Polyomavirus—a Brief Review of Recent Knowledge*

The hamster polyomavirus (HaPV) was first described in 1967 as a virus associated with skin epithelioma of the Syrian hamster. The tumors appear spontaneously in a hamster colony bred in Berlin-Buch (HaB). Virus particles isolated from skin epitheliomas cause lymphoma and leukemia when injected into newborn hamsters from a distinct colony bred in Potsdam, Germany (HaP). The viral genome has been totally sequenced and the overall genetic organization establishes HaPV as a member of the polyomaviruses. HaPV is a second example of an middle T (MT) antigen encoding polyomavirus and nucleotide sequence homologies designates the mouse polyomavirus (Py) as the closest relative.Lymphomas induced by HaPV in HaP hamsters do not contain virus particles but instead accumulate different amounts of nonrandomly deleted free and/or integrated viral genomes. Transgenic mice produced by microinjection of HaPV DNA into the pronucleus of fertilized eggs of Gat: NMRI mice developed both, epitheliomas and lymphomas. Both tumor types contain extrachromosomal DNA.HaPV DNA was found to replicate in hamster lymphoid and fibroblast cell lines. Fully reproductive cycles could be detected only in GD36 lymphoblastic leukemia cells.HaPV carries the full transforming properties of a polyomavirus in vitro. Immortalization of primary rat cells is essentially carried out by the HaPV large T (LT) antigen and coexpression of HaPV MT and HaPV small T (ST) antigen is required for full transformation of rat fibroblasts. The preferential binding of HaPV MT to c-Fyn, a Src family kinase, has been proposed as a mechanism leading to lymphoid malignancies.Heterologous expression of HaPV-VP1 allowed the formation of virus like particles (VLPs) resembling HaPV particles. The high flexibility of HaPV-VP1 for insertion of foreign peptides offers a broad range of potential applications, especially in vaccine development.     

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.     

An immunodominant, cross-reactive B-cell epitope region is located at the C-terminal part of the hamster polyomavirus major capsid protein VP1

The VP1 represents the major capsid protein of the hamster polyomavirus (HaPV). Here we describe the mapping of epitopes along the VP1 using Escherichia coli-expressed VP1-dihydrofolate reductase (DHFR) fusion proteins and PepScan analysis. By use of DHFR fusion proteins an immunodominant region was localized in the C-terminal part of VP1 between amino acids 320-384. Further epitopes are located in the regions amino acids 1-133 and amino acids 133-320, respectively. There were no obvious differences in the reactivity between sera of tumor-bearing and papilloma-free naturally HaPV-infected hamsters. In contrast, PepScan analysis revealed linear epitopes in the regions amino acids 79-97 and amino acids 353-367 for tumor-bearing animals and amino acids 101-113 and amino acids 165-179 for papilloma-free animals. The region between amino acids 320-384 of HaPV-VP1 was found to be involved in cross-reactivity of VP1 from HaPV and other polyomaviruses. Previously we have demonstrated that heterologous expression of HaPV-VP1 allowed the formation of virus-like particles (VLPs). From epitope mapping data and structural predictions it has been suggested that HaPV-VP1-VLPs may tolerate foreign peptides in the region amino acids 81-88 and the C-terminal part of VP1.     

Complete DNA sequence of lymphotropic papovavirus: prototype of a new species of the polyomavirus genus

Lymphotropic papovavirus (LPV) is a new member of the polyomavirus genus. Its host range in vitro is restricted to transformed cells of B-lymphocyte origin. Here the complete 5270-bp DNA sequence of LPV is presented. The LPV early region can encode a large T and a small t antigen but no middle T antigen and the late region can encode the three structural proteins VP1, VP2, and VP3. Based on sequence conservation of shared proteins LPV is equally related to both mouse polyomavirus (Py) and simian virus 40 (SV40) and represents a new distinct species of the polyomavirus genus. Sequence comparisons of LPV, SV40, and Py point out essential conserved sequence features of the polyomavirus genus more clearly than the comparison of only SV40 and Py. The most conserved proteins are VP1 with 42% and large T antigen with 28% of the amino acids conserved among the three viruses. Although least conserved the noncoding DNA sequences of LPV show significant homologies both to SV40 and Py (origin of viral DNA replication and putative early promoter). A 63-bp tandem repeat at the late side of the replication origin possibly represents a LPV enhancer element.     

Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells

As polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation. The hDCs stimulated by HaPyV- or MPyV-derived VLPs readily produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro. The highest frequencies of activated T cells were again observed after pulsing with HaPyV- and MPyV-derived VLPs. Monocyte-derived hDCs both bound and internalized the various tested polyomavirus VP1-derived VLPs with different levels of efficiency, partially explaining their individual maturation potentials. In conclusion, our data suggest a high variability in uptake of polyomavirus-derived VLPs and potency to induce hDC maturation.     

Serological cross‐reactivity between human polyomaviruses

Until 2006, BKPyV and JCPyV were the only known human polyomaviruses. A third polyomavirus, simian virus 40 whose natural host is the macaque was accidently introduced into man because of contaminated poliovirus vaccines, although there is epidemiological evidence that SV40 may be transmitted between man independently from contaminated vaccines. Since 2007, 10 new human polyomaviruses have been identified: KIPyV, WUPyV, Merkel cell polyomavirus, trichodysplasia spinulosa‐associated polyomavirus, and human polyomaviruses 6, 7, 9, 10, STL, and 12. Moreover, the DNA of the monkey lymphotropic polyomavirus has been amplified from human peripheral blood. Seroepidemiological studies frequently based on the presence of antibodies against the major capsid protein VP1 or virus‐like particles indicate that most human adults have been exposed to many, if not all, human polyomaviruses. However, because of the high amino acid sequence identity between VP1 of some human polyomaviruses, cross‐reactivity of antibodies is occasionally observed. In addition, human sera possess reactivity against VP1 of polyomaviruses from other species, suggesting serological cross‐reaction with known or closely related, yet unidentified human polyomaviruses and/or the possibility of zoonotic transmission. Thus, current serological results should be interpreted with caution, and controls excluding cross‐reactivity with other polyomaviruses are required. Copyright © 2013 John Wiley & Sons, Ltd.     

The hamster papovavirus: evolutionary relationships with other polyomaviruses

The hamster papovavirus (HaPV) is a polyoma virus with a restricted tumor spectrum. It is actively replicated in hair follicle tumors arising spontaneously in young Syrian hamsters. It can also induce lymphomas and leukemias in newborn hamsters. The complete nucleotide sequence of a cloned HaPV has been established recently. This report presents a comparison of this sequence with other polyomavirus genomes (polyoma, SV40, BKV, LPV) by matrix dot analysis and electron microscopy heteroduplex mapping. The results demonstrate a close relationship between the HaPV and the murine polyoma virus and designate the LPV as the closest relative among the primate polyomaviruses.     

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped + ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.     

Capsid assembly and DNA packaging in herpes simplex virus

The genome of HSV-1 contains 80–85 open reading frames. Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion. The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core. The capsid is an icosahedral shell whose major morphological features are 162 capsomers. It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26. VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers. In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a. DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins. Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques. Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly. More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids. The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads. This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging. © 1997 John Wiley & Sons Ltd.     

Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.     

Sequence homology between polyoma virus, simian virus 40, and a papilloma-producing virus from a Syrian hamster: evidences for highly conserved sequences

Sequence homology between the genomes of a hamster papovavirus (HaPV), polyoma virus (Py), and Simian virus 40 (SV40) has been studied by filter hybridization and electron microscopy under conditions of varying stringency. Hybrids between the HaPV and SV40 DNAs could be demonstrated only under nonstringent conditions. The region of highest homology was mapped in the early region of the SV40 genome. Extensive homology was detected between the genomes of HaPV and Py under stringent hybridization conditions, indicating at least 80% base matching in the regions of strongest sequence homology. These sequences were localized within both the early region and the late region of the Py genome. The homologous DNA segments mapped in the Py and the SV40 genomes are among the most strongly conserved regions in the polyoma (miopapova)-virus group.     

Analysis of hamster lymphomas for the presence of hamster papovavirus DNA

The hamster papovavirus (HaPV) is a polyomavirus isolated from skin epitheliomas arising spontaneously in young Syrian hamsters. It can induce lymphomas and leukaemias in newborn hamsters. Although no virus particles are detectable by electron microscopy, high amounts of monomeric and oligomeric forms of extrachromosomal HaPV DNA molecules are found in the lymphoma cells. These molecules display deletions of about 300 nucleotides in length. Their role in the lymphoma induction is discussed.     

Generation of lymphoma-type variant hamster polyomavirus genomes in hamsters susceptible to lymphoma induction

Summary. The hamster polyomavirus (HaPV) induces either hair follicle epitheliomas or lymphomas in either Z3 or HaP respectively, Syrian hamsters. In the lymphomas specifically deleted “lymphoma-type” (lt) HaPV genomes are accumulated. In the present study the temporal pattern of generation of HaPV (lt) DNA was investigated in context of the development of lymphomas in neonatally infected HaP hamsters. The generation of HaPV (lt) DNA was first detectable during the postnatal phase of high level replication of viral DNA in hemopoietic organs (at 7 days post infection), thus clearly preceding the development of overt lymphoma. A variety of HaPV (lt) DNA species is generated in lymphoid cells, but usually only one of them is accumulated to high amounts in lymphoma cells. Furthermore, the pattern of HaPV (lt) and wild-type (wt) DNA was studied in normal and tumor tissues of tumor-bearing hamsters as well as in tumor-free hamsters. In tumor-bearing hamsters predominantly HaPV (lt) DNA species were found in the infected tissues, while HaPV (wt) DNA was detected rarely and only in tumor-free tissues. In contrast, in tissues of tumor-free hamsters HaPV (wt) DNA prevailed over HaPV (lt) DNA species. Received May, 22, 1996 Accepted August 19, 1996     

Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus

The DNA of a virus isolated from fledgling budgerigars, designated BFDV, was cloned and analyzed with regard to its relationship to the polyomavirus subgroup of the papovavirus family. Under relaxed conditions, the DNA of BFDV cross-hybridized with the DNAs of members of the polyomavirus subgroup, such as the mouse polyomavirus, the monkey viruses simian virus 40, stump-tailed macaque virus, and lymphotropic papovavirus, and the human viruses JCV and BKV. Under stringent conditions, however, no homologies could be detected. Furthermore, BFDV propagated in chicken embryo cells was antigenically related to the capsid antigen(s) of the other polyomaviruses. The virus was able to transform hamster embryo cells in vitro which is a typical feature of polyomaviruses. These data clearly indicate that BFDV is a new distinct member of the polyomavirus genus representing the first nonmammalian polyomavirus.     

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.     

Subunit interaction in B19 parvovirus empty capsids

Summary B19 parvovirus is a small single-stranded DNA virus with a genome that encodes only two structural proteins, designated VP1 and VP2. 60 copies of the structural proteins assemble into the viral capsid, with approximately 95% VP2 and 5% VP1. Recombinant empty capsids composed of VP2 alone or of VP2 and VP1 self-assemble into particles that are morphologically indistinguishable from full virions. Empty capsids containing both VP2 and VP1 elicit a strong neutralizing antibody response when used to immunize rabbits. Capsids containing only VP2 are similarly antigenic but elicit only weak neutralizing activity. We performed fine structure epitope mapping by measuring the reactivity of antisera raised against capsids composed of VP2 and VP1 or VP2 alone against 85 overlapping peptides spanning the sequence of the two structural proteins. A profile of the antigenic difference between empty capsids with and without VP1 was produced from the resulting data. This profile divided the sequence of the structural proteins into four regions that correlated well with expected viral structures. Thus, the addition of a small number of VP1 residues altered the antigenicity of the entire capsid. The major area of enhanced antigenicity is homologous to the spike of canine parvovirus, an area known to contain both neutralizing and host-range determinants. Our data are consistent with a model in which the unique region of VP1 is necessary for the virus to assume its mature capsid conformation.     

Hamster polyomavirus-encoded proteins: Gene cloning,heterologous expression and immunoreactivity

The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain. Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA. To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as -galactosidase-, TrpE-and dihydrofolate reductase-fusion proteins or as non-fused proteins. Recombinant plasmids carried segments of all putative early and late HaPV proteins. The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti--galactosidase antibodies and sera of HaPV-infected hamsters.Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals. HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3. The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera. Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.     

Methylation of the polyomavirus major capsid protein VP1

Polyomavirus VP1 has been shown to be modified by phosphorylation, sulfation, acetylation and hydroxylation. The major capsid protein VP1 is now shown to be modified by methylation. Addition of cycloheximide to infected cultures followed by addition of [³H-methyl]- -methionine and subsequent immunoprecipitation, SDS-PAGE and fluorography revealed methylation occurring on VP1. Amino acid analysis of [³H]-methyl]- -methionine-labelled polyomavirus VP1 by two-dimensional paper chromatography and HPLC of the acid-hydrolyzed protein revealed the presence of ³H-labelled trimethyllysine and monomethylarginine.     

Hepatitis A virus subviral particles: purification, accumulation, and relative infectivity of virions, provirions and procapsids

Summary.  Virus-specific particles were isolated from hepatitis A virus (HAV)-infected cells and the role of each particle type in the replicative cycle assessed. Mature virions, provirions (immature virions) and empty capsids (procapsids) were detected in cell lysates, and both virions and provirions were found in the culture supernatant. Particle types were separated by isopycnic caesium chloride gradient- or linear sucrose density gradient-ultracentrifugation, and their capsid proteins characterised. Virions, provirions and procapsids containing both VP1 and varying levels of the VP1 precursor protein PX were found, suggesting that trimming of PX is not essential for particle formation. Provirions (containing VP0) and virions (containing VP2) could not be clearly separated with these techniques, but sucrose gradients allowed greater separation of particle pools with distinct VP0 contents and specific infectivities which could be used for further studies of the biological role of VP0 cleavage. Virions, with a higher sedimentation coefficient and buoyant density presumably reflecting a more compact structure, had a higher relative infectivity when compared to provirions. HAV-infected cells therefore contain a heterogenous mixture of RNA-containing viral particles with characteristics between those of true provirions and virions, but all such particles are released from the cell and can participate in further rounds of infection. Received April 8, 1997 Accepted July 9, 1997