A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

Abstract

19Following internalisation viruses have to escape the endocytic pathway and deliver their 20 genomes to initiate replication. Members of the Polyomaviridae transit through the 21 endolysosomal network and through the endoplasmic reticulum (ER), from which heavily 22 degraded capsids escape into the cytoplasm prior to nuclear entry. Acidification of 23 endosomes and ER entry are essential in the lifecycle of polyomaviruses, however many 24 mechanistic requirements are yet to be elucidated. Alteration of endocytic pH relies upon the 25 activity of ion channels. Using two polyomaviruses with differing capsid architecture, namely 26 Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we firstly describe methods 27 to rapidly quantify infection using an IncuCyte ZOOM instrument, prior to investigating the 28 role of K + and Ca 2+ channels during early stages of infection. Broad spectrum inhibitors 29 identified that MCPyV, but not SV40, is sensitive to K + channel modulation. In contrast, both 30 viruses are restricted by the broad spectrum Ca 2+ channel inhibitor verapamil, however 31 specific targeting of transient or long lasting Ca 2+ channel subfamilies had no detrimental 32 effect. Further investigation revealed that tetrandrine blockage of two-pore channels (TPCs), 33the activity of which is essential for endolysosomal-ER fusion, ablates infectivity of both 34 MCPyV and SV40 by preventing disassembly of the capsid, which is required for the 35 exposure of minor capsid protein nuclear signals necessary for nuclear transport. This study 36 therefore identifies a novel target to restrict the entry of polyomaviruses. 37 IMPORTANCE 38Polyomaviruses establish ubiquitous, asymptomatic infection in their host. However, in the 39 immunocompromised these viruses can cause a range of potentially fatal diseases. Through 40 the use of SV40 and MCPyV, two polyomaviruses with different capsid organisation, we 41 have investigated the role of ion channels during infection. Here, we show that Ca 2+ channel 42 activity is essential for both polyomaviruses and that MCPyV is also sensitive to K + channel 43 blockage, highlighting new mechanistic requirements of ion channels during polyomavirus 44 infection. In particular, tetrandrine blockage of endolysosomal-ER fusion is highlighted as an 45 essential modulator of both SV40 and MCPyV. Given that the role of ion channels in disease 46 have been well characterised, there is a large panel of clinically available therapeutics that Polyomaviruses (PyVs) are small double stranded DNA viruses that establish persistent 51 infections in their hosts. Whilst infections are generally asymptomatic, PyVs can cause 52 severe disease in the immunosuppressed. Common examples include BKPyV-associated 53 nephropathy and haemorrhagic cystitis, and JCPyV-induced progressive multifocal 54 leukoencephalopathy (PML) (1-3).55 In ~80% of Merkel cell carcinoma (MCC) cases, Merkel cell PyV (MCPyV) infection, clonal 56 integration and UV-mediated mutation of the viral genome occur prior to tumour c...

Pharmacological hypothesis: TPC2 antagonist tetrandrine as a potential therapeutic agent for COVID‐19

“…TPCs are widely expressed throughout the body 18 and have been implicated as key components in many physiological and pathological processes, including infection and immunity (reviewed in 19). Recently, TPCs have been implicated in Middle East Respiratory Syndrome‐Coronavirus (MERS‐CoV), 20 Ebola virus (EBOV), 9 and Merkel cell polyoma virus (MCPyV) 21 infection as key drug targets.…”

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection

“…Ion channels are emerging as key factors required during virus replicative cycles, examples including the roles for potassium (K + ) channels in Bunyamwera virus (BUNV) infection 9 10 ; two-pore calcium (Ca 2+ ) channels in Ebola virus (EBOV) infection 11 and chloride (Cl − ) channels in chikungunya virus (CHIKV), hepatitis C virus (HCV), BK polyomavirus and Merkel cell polyomavirus infection. [12][13][14][15] Cl − channels are diverse families of proteins that regulate cell excitability and fluid and osmolyte secretion in lung epithelial cells. Over 40 genes are linked to Cl − conductances which can be categorised into voltage-gated Cl − channels (ClC), ligand-gated Clchannels, cystic fibrosis transmembrane conductance regulator (CFTR), volume-regulated anion channels and Ca 2+ -activated Cl − channel (CaCC) families.…”

Tetrandrine as a Therapeutic Agent for COVID-19

“…Two-pore channels (TPCs) are intracellular calcium/cation channels located in the membranes of host endolysosomal compartments which SARS-CoV-2, the virus causing COVID-19 (and several other viruses) depend upon for egress from these organelles and replication. TPC2 has already been implicated in Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) [9], Ebola virus (EBOV) [10], and Merkel cell polyoma virus (MCPyV) [11] infection as a key drug target.…”