Virus cell surface binding and entry

Polyomaviruses bind various cell surface receptors before entering the cell. BKV, MuPyV and SV40 use different ganglioside receptors (Tsai et al. 2003; Low et al. 2006) while JCV has preferential binding for serotonin receptor

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(Elphick et al. 2004). Virus entry mechanism is determined by attachment receptors, for instance, BKV, MuPyV and SV40 undergo caveolar endocytosis (Norkin et al. 2002; Eash et al. 2004; Gilbert and Benjamin 2004). Caveolar endocytosis depends on caveolin proteins and formation of flask-shape invaginations in the cell membrane called caveolae (Pelkmans and Helenius 2002), in which gangliosides accumulate (Sapp and Day 2009). Caveolae never join lysosomes and therefore viruses using this internalisation route avoid lysosomal degradation (Kiss and Botos 2009). Following attachment, SV40 virions move to caveolin 1-containing caveolae which will then depart from cell membrane and join pre-existing pH-neutral and cholesterol-rich stationary organelles called caveosomes (Pelkmans et

al. 2001). A few hours after SV40 entry, caveosomes transform into long

tubular membrane structures which no longer contain caveolin-1, detach from caveosomes and transport virions to smooth endoplasmic reticulum (ER) where SV40 accumulates and disassembles (Pelkmans et al. 2001; Norkin et

al. 2002). A more recent study revealed that SV40 internalisation still occurs

in caveolin-deficient cells, suggesting that caveolin-1 is not essential for membrane-to-ER transport (Stergiou et al. 2013). The authors also demonstrated that SV40 binding of integrins, a type of bidirectional signalling and adhesion receptor (Hynes 2002), triggers recruitment of PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) to the membrane, leading to de-phosphorylation of Ezrin, a member of ezrin-radixin-moesin (ERM) family (Stergiou et al. 2013), which are important regulators of membrane domains (Fehon et al. 2010). Stergiou et al. suggest that Ezrin de- phosphorylation has several consequences including disconnection of

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cortical actin from plasma membrane and membrane tubulation, both of which are required for SV40 internalisation (Ewers et al. 2010; Stergiou et al. 2013). Inhibition of actin filaments with jasplakinolide resulted in reduced BKV infectivity, suggesting that BKV too utilises the actin cytoskeleton for virus infection (Eash and Atwood 2005).

Unlike other polyomaviruses, JCV utilises clathrin-dependent endocytosis (Pho et al. 2000), an endocytosis mode which involves formation of clathrin- coated pits and various adaptor proteins such as eps15 (epidermal growth factor receptor pathway substrate clone 15), adaptor protein complex 1 (AP1), AP2 and AP180 (Motley et al. 2003; Ungewickell and Hinrichsen 2007; Schelhaas 2010). Studies have shown that JCV infectious entry to glial cells requires clathrin, eps15 and activated tyrosine kinase signalling (Querbes et al. 2004). In addition, JCV infection of glial cells activates mitogen-activated protein kinases 1 (MAPK1) and MAPK2, probably to facilitate efficient virus internalisation, cellular trafficking and replication (Querbes et al. 2004).

MCV attachment and entry is comprised of two steps in which the virus sequentially binds to glycosaminoglycans and sialylated oligosaccharides before cell entry. The initial attachment occurs via binding of MCV capsid protein VP1 to heparan sulfate (HS) and chondroitin sulfate (CS) (Schowalter

et al. 2011), then VP1 via its apical shallow binding site binds Neu5Ac-α2,3-

Gal, a linear motif of N-acetyl neuraminic acid (Neu et al. 2012). Interestingly, while the sialylated oligosaccharides are not required for the initial entry they are indispensable for the follow-up infectious entry of the virus (Schowalter et

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1.4.2.2. Virus replication

Like other polyomaviruses, MCV requires the expression of LT to initiate replication and is largely dependent on the host cell machinery for successful transcription and replication of its genome. Virus replication is a delicately coordinated process in polyomaviruses, which begins with transcription of the early region upon nuclear entry of the genome, followed by oligomerisation of LT antigen in the form of hexamers (DeCaprio and Garcea 2013). In SV40, binding of ATP to LT triggers interaction between two LT hexamers and ORI followed by movement of hexamers in opposite directions, which combined with helicase activity unwinds the viral genome and advances bi-directional genome replication (Borowiec and Hurwitz 1988; Wessel et al. 1992). As mentioned in section 1.4.1, a minimum core region of 71bp in the MCV ORI has been reported as sufficient for initiation of the virus replication. This core region is composed of ten G(A/G)GGC pentamer (P1 to P10) repeats, as recognition sites for LT OBD, and flanking AT-rich sites. As can be seen in Figure 1.11 B, LT OBD binds the central P1, P2 and P4 sites. In fact, these three sites, along with P7, are indispensable for origin replication, although P7 is located further and outside the origin centre (Harrison et al. 2011). OBD recognition of the essential pentanucleotides leads to binding of LT hexamers in an apex-to-apex orientation while wrapping around the dsDNA genome (Figure 1.11 C). It has been suggested that the flanking AT-rich sequences (Figure 1.11 A) allow for easier melting of the core origin and hence facilitates replication initiation (Kwun et al. 2009).

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Figure 1.11. The minimum core region of MCV origin of replication required

for efficient virus replication (A), schematic presentation of LT OBD binding to the central pentanucleotides P1, P2 and P4 based on X-ray crystallography (B) and LT hexamers binding, genome unwinding and replication initiation (C). The pentanucleotide repeats (P) are boxed and essential Ps are highlighted in red. Adapted from (Harrison et al. 2011) and (DeCaprio and Garcea 2013).

Other polyomaviruses such as BKV, JCV and SV40 have the same origin pentamer repeats, but unlike MCV, these are in a symmetrical arrangement with different inter-pentamer spacing. Moreover, while in BKV, JCV and SV40 the indispensable P1-P4 repeats are juxtaposed and show no overlapping, in MCV the essential P1, P2, P4 and P7 are separated by the second AT-rich site and the dispensable overlapped P5/P6 (Figure 1.11 A). The importance of such structural distinctions in the replication origins of polyomaviruses has

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remained unexplained to date, however, what is clear is that despite perfect identity of the binding sites and noticeable identities among OBDs in MCV and SV40, cross replication does not occur by heterologous LTs (Harrison et

al. 2011).

In tumour derived MCV, replication capacity is lost following a single point mutation in essential pentanucleotides (Kwun et al. 2009). Furthermore, consistent with the observation in MCV-positive MCC, loss of OBD and helicase domains renders MCV non-replicative (Shuda et al. 2008). Interestingly, whilst LT alone is sufficient for virus replication, efficient genome replication is achieved only in the presence of both sT and LT. In contrast, 57kT does not improve replication capacity of LT, and sT or 57kT alone cannot initiate or drive replication. As mentioned earlier, LT shares the first exon, and therefore some domains such as DnaJ with sT (Figure 1.9). DnaJ is a conserved motif in polyomaviruses and is required for efficient replication in SV40 (Sheng et al. 1997). It has been shown that, despite sharing the DnaJ domain, replication efficiency is significantly reduced following mutation in LT DnaJ whereas mutation of sT DnaJ does not alter the enhancing capacity of sT in LT-driven replication (Kwun et al. 2009). In contrast, a mutated sT PP2A domain does eliminate the replication enhancing capacity of sT, suggesting a role for sT and PP2A in efficient LT replication (Kwun et al. 2009). However, a later study revealed via domain mutation that any such effect is not mediated by sT PP2A, but rather by a domain located on the opposite side of PP2A termed LSD or LT-stabilisation domain (Kwun et al. 2013).

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SCF (SKP1, CUL1 and F-box containing complex) is a conserved ubiquitin ligase complex and FBW7 (F-box and WD repeat domain-containing 7) is a subunit of SCF which regulates proto-oncogenes and proteins involved in cell division, growth and differentiation (Welcker and Clurman 2008). FBW7 dysregulation and mutation has been shown in several malignancies including T-cell acute lymphoblastic leukaemia/lymphoma (Maser et al. 2007), breast and colorectal cancers (Wood et al. 2007), cholangiocarcinomas and tumours of endometrium and stomach (Akhoondi

et al. 2007). These studies suggest that FBW7 is an important tumour

suppressor which promotes degradation of proto-oncogenes in a phosphorylation-dependent manner (Koepp et al. 2001; Strohmaier et al. 2001; Yada et al. 2004). Strikingly, LSD inhibits SCFFBW7-mediated proteasomal degradation of LT, hence indirectly enhancing MCV replication by promoting the accumulation of LT. Interestingly, sT-mediated SCFFBW7 inhibition also reduces turnover of cellular proto-oncogenes c-Myc and Cyclin E (Kwun et al. 2013), a process which can promote cell transformation.

T antigen-mediated hijacking of cellular machinery in favour of virus replication is not limited to SCFFBW7. DNA damage response (DDR) machinery governs the recognition and repair of DNA damage in the cell. Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases are two important components of DDR, which can mediate cell cycle arrest and apoptosis upon activation (Ciccia and Elledge 2010; Tsang et al. 2014). MCV LT has been shown to interact and co-localise with DDR elements in the nucleus in an ORI and replication-dependent manner. Furthermore, inhibition of DDR and elimination of ATM and ATR abolishes

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MCV genome replication (Tsang et al. 2014), indicating the importance of DDR in MCV life cycle. This crucial role for DDR in the replication of polyomaviruses has also been described in SV40 (Sowd et al. 2013), MuPyV (Dahl et al. 2005), JCV (Orba et al. 2010) and BKV (Jiang et al. 2012). Another cellular protein contributing to MCV replication is bromodomain and extra terminal domain (BET) family member, Bromodomain-containing protein 4 or Brd4. Brd4 has been implicated in cell cycle progression to S phase (Mochizuki et al. 2008) and cancer (French et al. 2001; Zuber et al. 2011). It has also been associated with oncogenic viruses such as EBV, KSHV and papillomaviruses (Ottinger et al. 2006; Wu et al. 2006; Lin et al. 2008). Replication factor C (RFC) is a chaperone-like protein complex, which upon recognition of template-primer 3’ ends, recruits and loads Proliferating cell nuclear antigen (PCNA) to DNA. PCNA is a DNA clamp which encircles and slides along DNA like a ring while acting as a tether for DNA polymerases (Moldovan et al. 2007). Wang et al. showed both in vivo and in

vitro that MCV LT interacts with Brd4 at MCV replication sites and that this

interaction is required for virus replication, since knockdown of Brd4 markedly reduces virus replication. Furthermore, similar to SV40 (Wold and Kelly 1988), MCV LT recruits replication protein A 70 (RPA70) to stabilise the nascent unwound viral ssDNA and prevent it from reannealing (Wold 1997). Directly interacting with LT, Brd4 then recruits RFC, PCNA and DNA polymerase delta to replication sites and facilitates viral DNA elongation (Wang et al. 2012).

Unlike the proteins described so far, hVam6p, which is a cellular vacuolar sorting protein, reduces MCV replication. hVam6p is part of HOPS or

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homotypic fusion and protein sorting complex which modulates lysosome and late endosome fusion (Caplan et al. 2001). Direct interaction of MCV LT and hVam6p via a site near LT pRb-binding domain (Figure 1.9) re-localises hVam6p to the nucleus in an LT NLS-dependent manner and prevents lysosome clustering (Liu et al. 2011). While overexpression of hVam6p dramatically reduces MCV replication, loss of LT-hVam6p interaction enhances virion production by up to 6-fold (Feng et al. 2011). In addition, hVam6p isoforms have been shown to be involved in mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β) signalling pathways, however, the interaction between MCV LT and hVam6p does not seem to affect these (Liu et al. 2011). Although further studies are required with regards to MCV dependence on hVam6p for cell growth and proliferation, current data suggest that MCV-driven tumourigenesis is not mediated by this cellular protein since mutation-driven loss of LT-hVam6p interaction does not affect cell viability or proliferation (Feng et al. 2011). Therefore, from a cellular point of view, the LT-hVam6p interaction might be important in the context of innate immunity, i.e. preventing persistent infection via disruption of MCV replication (Stakaityte et al. 2014). A second possibility is that MCV-mediated disruption of lysosome clustering might be important for virus uncoating or egress (Liu et al. 2011).