The discovery was published in the peer reviewed journal: Virologica Sinica.
https://www.virosin.org/article/doi/10.1016/j.virs.2022.06.001
. . . .
Emergence and re-emergence of infectious diseases of wildlife origin have led pre-emptive pathogen surveillances in animals to be a public health priority. Rodents and shrews are among the most numerically abundant vertebrate taxa and are known as natural hosts of important zoonotic viruses. Many surveillance programs focused more on RNA viruses. In comparison, much less is known about DNA viruses harbored by these small mammals.
To fill this knowledge gap, tissue specimens of 232 animals including 226 rodents, five shrews and one hedgehog were collected from 5 counties in Kenya and tested for the presence of DNA viruses belonging to 7 viral families by PCR. Diverse DNA sequences of adenoviruses, adeno-associated viruses, herpesviruses and polyomaviruses were detected. Phylogenetic analyses revealed that most of these viruses showed distinction from previously described viruses and formed new clusters.
Furthermore, this is the first report of the discovery and full-length genome characterization of a polyomavirus in Lemniscomys species. This novel polyomavirus, named LsPyV KY187, has less than 60% amino acid sequence identity to the most related Glis glis polyomavirus 1 and Sciurus carolinensis polyomavirus 1 in both large and small T-antigen proteins and thus can be putatively allocated to a novel species within Betapolyomavirus.
Our findings help us better understand the genetic diversity of DNA viruses in rodent and shrew populations in Kenya and provide new insights into the evolution of those DNA viruses in their small mammal reservoirs. It demonstrates the necessity of ongoing pathogen discovery studies targeting rodent-borne viruses in East Africa.
… AND
It should be noted that although the polyomaviruses identified is an animal virus, studies have shown that animal polyomaviruses when injected into other species including humans, can cause the rise of cancerous tumors.
https://www.frontiersin.org/articles/10.3389/fmicb.2022.834368/full
The Polyomavirus Family: Genome Organization
Polyomaviruses (PyVs) are a family of small, non-enveloped viruses that can infect fish, birds, and mammals, including humans (Moens et al., 2017a). Characteristic for PyVs is the circular double-stranded DNA genome of approximately 5.0 kbp that encodes two major regulatory proteins, the large tumor antigen (LT-ag) and the small tumor antigen (sT-ag), and at least two structural proteins (VP1 and VP2). The regulatory genes and structural genes are separated by a non-coding control region (NCCR) encompassing the origin of replication and the promoter/enhancer sequences (DeCaprio and Garcea, 2013; Moens et al., 2017b). The regulatory proteins are expressed early during infection and participate in viral replication and viral transcription, while the structural proteins, which are expressed later in the infection cycle, form the capsid (DeCaprio and Garcea, 2013; Baez et al., 2017; Moens et al., 2017b). LT-ag contains an origin binding domain (OBD) that binds tandem repeated 5′-GRGGC-3′ motifs in the NCCR and a helicase/ATPase domain in its C-terminus. The OBD and helicase/ATPase domain are required for viral genome replication (Borowiec et al., 1990). Many PyVs encode additional regulatory and structural proteins (e.g., middle tumor antigen or MT-ag, ALTO, VP3, VP4, agnoprotein) (Carter et al., 2013; Moens et al., 2017b; Saribas et al., 2019).
To date, fifteen PyVs have been isolated from different human specimens (Moens et al., 2020). The first human polyomaviruses (HPyVs), BKPyV, and JCPyV, were identified in 1971 (Gardner et al., 1971; Padgett et al., 1971). In 2007, two new HPyVs, Karolinska Institute Polyomavirus (KIPyV) and Washington University Polyomavirus (Allander et al., 2007) (WUPyV) (Gaynor et al., 2007), were detected, and in the following years, Merkel cell Polyomavirus (MCPyV) (Feng et al., 2008), HPyV6 (Schowalter et al., 2010), HPyV7 (Schowalter et al., 2010), Trichodisplasia spinulosa polyomavirus (TSPyV) (van der Meijden et al., 2010), HPyV9 (Sauvage et al., 2011; Scuda et al., 2011), HPyV10 and its variants Malawi polyomavirus (MWPyV) and Mexico polyomavirus (MXPyV), (Buck et al., 2012; Siebrasse et al., 2012; Yu et al., 2012), Saint Louis polyomavirus (STLPyV) (Lim et al., 2013), HPyV12 (Korup et al., 2013), New Jersey Polyomavirus (NJPyV) (Mishra et al., 2014), and Lyon IARC polyomavirus (LIPyV) (Gheit et al., 2017) have been described. Recently in 2019, Ondov et al. (2019) identified the Quebec polyomavirus (QPyV) sequences in the stool from one patient through the MinHash algorithm. Not all PyVs, originally isolated from human specimens, may be true HPyVs. HPyV12, first described in a human liver sample, was later shown to infect shrews as its natural host and was therefore reclassified as a Sorex araneus PyV (Sara-PyV1) by the International Committee on Taxonomy of Viruses (ICTV) (Gedvilaite et al., 2017). LIPyV DNA was first amplified from human skin but is most likely a feline PyV (Fahsbender et al., 2019; Li et al., 2021). LIPyV and QPyV have not yet been listed as an HPyV by the ICTV. QPyV DNA has been detected in urine from systemic lupus erythematosus patients, multiple sclerosis patients, and pregnant women, but more studies are required to confirm whether this is a genuine HPyV (Prezioso et al., 2021).
In this review we define novel human polyomaviruses (nHPyVs) as KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, STLPyV, HPyV12, LIPyV, and QPyV. Although HPyV12/Sara-PyV1 and LIPyV are not genuine HPyVs, and QPyV has not been classified as a HPyV, we will use the name HPyV12 and include HPyV12, LIPyV and QPyV as “nHPyVs”. Simian virus 40 (SV40) has also been detected in healthy and malignant samples from humans but is not considered a HPyV (Moens et al., 2017a). Because of its tremendous importance in understanding the oncogenic potentials of PyV, SV40 will be included as the prototype transforming PyV. Murine PyV (MPyV) and hamster Pyv (HaPyV) will also be discussed.
Seroprevalence of Human Polyomaviruses in the Healthy Population
Serological studies mainly based on the presence of HPyV VP1 antibodies detected by a VP1- or virus-like particle-based ELISA have demonstrated that HPyV infection is very common in healthy individuals. For most HPyVs, seroprevalence in the adult healthy population is > 70%, and most individuals are infected with more than one HPyV (Kean et al., 2009; Gossai et al., 2016; Kamminga et al., 2018). Seroprevalences for HPyV12, NJPyV-2013, and LIPyV are < 10% in all age categories tested, whereas another study reported a prevalence of 97% and 58% for HPyV12 and NJPyV-2013, respectively (Gaboriaud et al., 2018; Kamminga et al., 2018). The seroprevalence of QPyV has not been examined. A significant number of individuals acquire HPyVs already during their early life, which might become a requirement for the onset of a possibly associated disease or cancer later in the lifecycle (Kean et al., 2009; Gossai et al., 2016; Kamminga et al., 2018).
Human Polyomaviruses as Proven Causative Agents in Human Diseases
So far, six HPyVs are firmly associated with diseases. BKPyV can cause nephropathy and hemorrhagic cystitis in kidney and in hematopoietic stem cell transplants, respectively (Helle et al., 2017); JCPyV is a causative agent of progressive multifocal leukoencephalopathy (PML), primarily in HIV-positive patients (Cortese et al., 2021); TSPyV is linked to the rare skin disease trichodysplasia spinulosa (TS) (Kazem et al., 2013), and HPyV6 and HPyV7 are associated with pruritic rash (Klufah et al., 2021).
A human virus is considered a tumor virus when viral sequences or proteins are regularly and persistently present in tumors and there is epidemiological evidence that virus infection represents a major risk factor for cancer development. Moreover, it is demonstrated that the virus or specific virus genes can transform cells or induce tumors in suitable animal models and that transformation of cell and tumor induction in animals depends on continuous expression of viral protein(s) (zur Hausen, 2001). According to these criteria, IARC has classified the human viruses Epstein-Barr virus, Kaposi’s sarcoma-associated herpes virus, hepatitis B virus, hepatitis C virus, high-risk human papillomaviruses, human T-cell lymphotropic virus type 1, and human immunodeficiency virus as group 1 carcinogenic viruses (i.e., carcinogenic to humans) (Bouvard et al., 2009).
As for HPyV, only 3 members may be associated with cancer. Presently, MCPyV is the only HPyV considered to cause cancer in its host. Approximately 80% of Merkel cell carcinomas (MCC) are positive for the MCPyV genome, which is typically integrated and encodes a truncated form of LT-ag (Chang and Moore, 2012). As early as in 2012, MCPyV has been categorized as a group 2A carcinogen by the International Agency for Research on Cancer (IARC) (Bouvard et al., 2012). MCPyV has also been discovered in non-neoplastic B cells and neoplastic B cells, thus suggesting a role in B-cell neoplasia (Pantulu et al., 2010; Teman et al., 2011; Imajoh et al., 2012). BKPyV and JCPyV have been suspected to be involved in renal, prostate, colon and brain cancer (Fioriti et al., 2005; Niv et al., 2005; White et al., 2005; Delbue et al., 2014, 2017; Keller et al., 2015; Starrett and Buck, 2019; Ahye et al., 2020). Both viruses are classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (Bouvard et al., 2012).
The aim of this review is to provide an overview of the pro and contra evidence that argues for or against a possible role of nHPyVs in cancer. The implication of MCPyV in cancer has been extensively elaborated on in recent reviews (Chang and Moore, 2012; Becker et al., 2017; Csoboz et al., 2020; Pietropaolo et al., 2020; DeCaprio, 2021; Krump and You, 2021), so this virus will therefore only briefly be considered in this review. Although an emerging role for HPyV6 and HPyV7 in cancer was recently described in an excellent review (Klufah et al., 2021), we will include these two viruses.
Conserved Domains in the Early Proteins of Novel Human Polyomaviruses That May Contribute to Oncogenesis
Transformation Functions of the Early Proteins of SV40, MPyV, and MCPyV
The story of PyVs begins in the 1950s when Ludwik Gross showed that a filterable agent from leukemia extract from the inbred Ak mice, a strain that spontaneously developed leukemia, could cause tumors of the parotid when injected in newborn non-leukemic C3H mice. Hence, he named the virus parotid tumor virus. However, some mice developed additional tumors, and this was confirmed by work by Sarah Stewart and Bernice Eddy, who then renamed the virus SE polyomavirus reviewed in Morgan (2014). Our understanding of how PyV causes tumors came with the isolation of another polyomavirus from rhesus macaque origin, SV40. This virus was discovered in 1960 as a contaminant of poliovirus vaccines (Sweet and Hilleman, 1960; Hilleman, 1998). SV40 can transform cells, including human cells, induce tumors in animal models (but not its natural host), and can be detected in human tumors. As previously mentioned, a role of MCPyV in human cancer was demonstrated in 2008 (Feng et al., 2008).
The major oncoprotein of SV40 is LT which exerts is transforming functions by interfering with the tumor suppressors retinoblastoma protein and p53. SV40 LT has also been shown to bind the mitotic spindle checkpoint kinase Bub1, the E3 ubiquitin kinase Cul7, the insulin receptor substrate 1, and the DNA repair enzyme Nbs1. These interactions contribute to the oncogenic properties of SV40 LT [reviewed in Cheng et al. (2009)]. SV40 sT alone cannot transform cells but cooperates with LT. sT exerts its transforming role mostly by interacting with protein phosphatase 2A (PP2A) [reviewed in Cheng et al. (2009)]. The major transforming ability of MPyV depends on its middle T-antigen (MT) and sT. Both can impede the function of PP2A, whereas MT can bind and activate the tyrosine kinase Src (reviewed in Cheng et al. (2009)]. MCPyV-positive Merkel cell carcinomas express a truncated LT that can interact with retinoblastoma proteins, but not p53. In vitro studies have suggested that sT may be the major oncogenic protein (Chang and Moore, 2012; Pietropaolo et al., 2020; Ahmed et al., 2021). The functional motifs involved in transformation by LT, sT and MT will be described in more detail and their presence in the corresponding proteins of the nHPyVs will be discussed in the next sections.
Functional Domains in the LT of Novel Human Polyomaviruses That May Be Involved in Oncogenic Processes
Cell culture studies with temperature sensitive mutants demonstrated that the oncogenic potential of SV40 primarily depends on its LT-ag and this was later confirmed by animal studies (Noonan and Butel, 1978; Sáenz Robles and Pipas, 2009; Hudson and Colvin, 2016). LT-ag of BKPyV and JCPyV are also strongly oncogenic in heterologous animal models (Small et al., 1986a,b; Dalrymple and Beemon, 1990; Noguchi et al., 2013; Del Valle and Khalili, 2021). SV40 LT-ag interferes with DNA repair, apoptosis, cellular transcription, protein degradation, telomerase activity, immune and inflammatory responses, and stimulate cell proliferation, angiogenesis, and cell migration. LT-ag of other PyVs such as mouse PyV (MPyV), BKPyV, and JCPyV have been shown to (at least partially) possess the same functions. The oncogenic potential of SV40 and other PyVs LT-ag predominantly depends on its ability to bind and impede the function of the tumor suppressor proteins p53 and retinoblastoma (Moens et al., 2007; Cheng et al., 2009; Topalis et al., 2013; Baez et al., 2017).
The retinoblastoma tumor suppressor family contains the proteins, pRb, p107, and p130, encoded by the RB1, RBL1, and RBL2 genes, respectively. The retinoblastoma proteins (RB) are key proteins in regulating G1 to the S phase cell cycle progression through their interaction with the E2F transcription factors family (Genovese et al., 2006; Dick and Rubin, 2013). The interference with RB’s function by LT-ag requires a direct interaction mediated by the RB-binding motif (or pRb pocket) LXCXE (Stubdal et al., 1997; Sullivan et al., 2000; Brown and Gallie, 2002). The psycho (PTYGTX9F) motif is also important for RB binding. Moreover, an intact DnaJ domain, located in the N-terminal end of LT-ag, is also involved.
The DnaJ domain contains the constant region 1 (CR1; LXXLL) and the Hsc70 binding motif HPDKGGD/N (Campbell et al., 1997; Srinivasan et al., 1997; Sullivan and Pipas, 2002). The binding of LT-ag to RB promotes the activation of E2F, resulting in expression of genes required for S phase progression. Hsc70 is a chaperone with weak ATPase activity that binds to the DnaJ motif HPDKNGN/D. The binding of Hsc70 to LT-ag increases the intrinsic ATPase activity of Hsc70, with this interaction helping to disrupt pRb proteins/E2F complexes (Sullivan et al., 2000; Garimella et al., 2006; Salma et al., 2007). The binding of SV40 LT-ag and JCPyV LT-ag to Hsc70 stimulates cell cycle progression, and influences viral DNA replication, transformation, viral and cellular promoter activity, as well as virion assembly [reviewed in Frisque et al. (2006), Sullivan and Pipas (2002)]. LT-ags of SV40, BKPyV, and JCPyV bind all three retinoblastoma proteins, and may explain LT-ag’s transforming properties in vitro and in vivo (Harris et al., 1996; White and Khalili, 2006).
The CR1, the Hsc70 binding motif, and the RB-binding motif seems to be conserved in the LT-ag of most nHPyVs. A possible interaction between RB and LT-ag was not examined for all nHPyVs. LT-ag of MWPyV was found to bind pRb, p107 and p130, but failed to increase expression of the E2F target genes CCNA (encoding cyclin A), CCNE (encoding cyclin E), and MYBL2 (encoding B-MYB), and to decrease pRb levels contrary to SV40 LT-ag (Berrios et al., 2015). LT-ags of WUPyV, HPyV6, HPyV7, and TSPyV were also found to interact with the family member pRb by co-immunoprecipitation assays with lysates of cells overexpressing LT-ag (Rozenblatt-Rosen et al., 2012; Wu et al., 2016a; Nako et al., 2020). The biological relevance of HPyV7 LT-ag and pRb interaction remains unknown as HPyV-7 LT-ag expression in thymic epithelial tumors did not correlate with the phosphorylation of pRb (Keijzers et al., 2015). TSPyV LT-ag clusters with the cell proliferation marker Ki-67 and with phosphorylated pRb in hair follicles of TS-affected patients, thus suggesting a role for TSPyV LT-ag in cell proliferation and a potential driver of papule and spicule formation, typical for trichodysplasia spinulosa (Kazem et al., 2014).
Another essential LT-ag interaction in PyVs-mediated tumorigenesis is that with p53, a tumor-suppressing protein that regulates the gene expression in response to stressful events, such as DNA damage, leading to cell apoptosis, cell cycle arrest, or senescence. p53’s function is usually deregulated in many cancer types (Muller and Vousden, 2013). The interaction of PyV LT-ag with p53 requires the C-terminal part of the protein, which also contains the helicase/ATPase domain. The interaction of LT-ag with p53 prevents p53 from binding to DNA, and represses the transactivation domain of p53 (Sheppard et al., 1999). During SV40 carcinogenesis, LT-ag binds and blocks p53 activity, thereby preventing apoptosis, cell cycle arrest, DNA repair and angiogenesis (Vogelstein et al., 2000; Levine and Oren, 2009). Kellogg and coworkers determined the percentage identity across the p53 domains of HPyVs BK, JC, KI, WU, MC, 6, 7, TS, 10, STL, and NJ with the SV40 LT-ag p53-interaction domain (Kellogg et al., 2021). BKPyV and JCPyV LT-ag, which have been shown to interact with p53, had the highest identity (67 and 69%, respectively). They also predicted that the interaction of SV40 LT-ag with p53 requires 13 residues: D402, W581, Y582, P584, V585, Q590, Q593, K600, D604, F607, L609, S610, and Y612. Only W581 and Q583 are conserved in MPyV LT-ag. Accordingly, MPyV LT-ag does not bind p53 (Qian and Wiman, 2000). There is a high conservation among these residues, with many identical or similar in LT-ag of HPyVs and MPyV and HaPyV (Figure 1 and Supplementary Figure 1). Their computational docking studies of p53 with the LT-ags of BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, MWPyV, STLPyV, and NJPyV supported the possibility of all LT-ags to bind p53. These findings predict the possibility of nHPyV LT-ags to interact with p53 in a manner similar to SV40 LT-ag. A direct interaction between the LT-ag of BKPyV and the LT-ag of JCPyV and p53 has been demonstrated (Shivakumar and Das, 1996; Staib et al., 1996). Less is known about the nHPyVs. Full-length MCPyV LT-ag fails to bind p53, whereas the truncated LT-ag form expressed in MCC cells lacks the C-terminal domain, and hence the p53-binding region (Cheng et al., 2013; Borchert et al., 2014). The TSPyV LT-ag expressed in HEK293 cells did not or only weakly bound to p53 (Wu et al., 2016a; Nako et al., 2020). The reciprocal co-immunoprecipitation with lysates of osteosarcoma U2OS cells ectopically expressing MWPyV LT-ag, an HPyV10 variant with > 95% nucleotide identity (Siebrasse et al., 2012), and p53 demonstrated an interaction between these two proteins (Berrios et al., 2015). However, compared to SV40 LT-ag, MWPyV LT-ag was less stable and could not stabilize p53, nor could MWPyV LT-ag promote the growth of human diploid fibroblast IMR-90 cells (Berrios et al., 2015). The rapid turn-over of MWPyV LT-ag compared to SV40 LT-ag may explain its inability to promote cell growth and its lack of oncogenic potential.
SV40 LT-ag can interact with additional cellular proteins, which may contribute to viral transformation (Cheng et al., 2009). One of these SV40 LT-ag-binding proteins is the mitotic checkpoint serine-threonine protein kinase Bub1 (Cotsiki et al., 2004; Hein et al., 2009). Impaired function of Bub1 leads to chromosomal instability, as observed in cells expressing SV40 LT-ag (Hu et al., 2013). Interaction with Bub1 requires SV40 LT-ag residues 88-98, which contain the WEQWW motif. The LT-ags of most nHPyVs contain the conserved motif WD/EXWW (Figure 1 and Supplementary Figure 1). The oncogenic MCPyV, MPyV, and HaPyV lack the Bub1-binding motif (Figure 1), and LT-ag of the HPyV6 isolate H6-cg-A2f.11 has the mutated WGQWW motif, suggesting that the Bub1:LT-ag interaction may not be absolutely required for HPyVs’ transformation in vivo (Torres et al., 2018).
Another mechanism by which SV40 LT-ag can induce transformation is through interaction with the cellular protein Cul7, an E3 ubiquitin ligase (Kohrman and Imperiale, 1992; Ali et al., 2004). Binding requires residues 69-81 (AHQPDFGGFWDAT) and 98-102 (FNEEN). Mutation of F98 diminishes Cul7 binding, while deletion of amino acids 68-83 abolishes it (Cavender and Tevethia, 2016). Cul7 binding to SV40 LT-ag was shown to play a role in transformation because mouse embryonal fibroblasts (MEFs) expressing non-Cul7 binding LT-ag mutants were unable to form colonies in soft agar, while wild-type expression cells were able to do so (Ali et al., 2004). The FNEEN motif is partially conserved in the nHPyV KI, WU, 6, 7, 10, TS, STL, and Q, whereas only the BKPyV, JCPyV, KIPyV, and WUPyV LT-ags show reminiscent identity with the SV40 LT-ag 69-81 amino acid sequence (Figure 1 and Supplementary Figure 1). The interaction between LT-ags from the nHPyV and Cul7 has not been studied, although the low sequence identity may indicate that no such binding occurs.
Insulin receptor substrate 1 (IRS1) is a component of the insulin-like growth factor (IGF-I) signaling pathway that transduces signals from the IGF-I receptor (IGF-IR). SV40 LT-ag was found to bind IRS1 (Fei et al., 1995). The biological importance of the IRS-LT-ag interaction in transformation is underscored by the observation that SV40 LT-ag is unable to transform IGF-IR–/– MEFs, whereas LT-ag lacking the N-terminal 250 amino acids fails to bind IRS1 and to transform IGF-IR–/– MEFs overexpressing IRS1 (Sell et al., 1993; Fei et al., 1995). The E107K mutation in the pRb motif LFCYE abrogated binding of SV40 LT-ag to IRS. Despite conservation of this residue, it is not known whether LT-ag of the other HPyVs can bind IRS1, with the exception of JCPyV LT-ag. JCPyV LT-ag was found to bind IRS1, resulting in nuclear translocation of IRS-1. IRS-1 could then bind Rad51 and inhibit homologous recombination DNA repair (Lassak et al., 2002; Reiss et al., 2006).
The interaction of SV40 LT-ag with the DNA repair enzyme Nbs1 disrupts DNA replication control and has been suggested to help immortalization of cells. This interaction is mediated by the DNA binding domain of SV40 LT-ag (Lanson et al., 2000; Wu et al., 2004). It is not known whether LT-ags of other HPyVs can associate with Nbs1.
