Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

Abstract

Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. The other human coronaviruses (HCoV)-HCoV-229E, HCoV-OC43, NL63, and HKU1-are collectively responsible for about 10 to 30% of common colds. Generally harmless and selflimiting, these HCoV are also implicated in severe clinical outcomes, particularly in immunocompromised individuals, infants, and the elderly (3). Other coronaviruses cause considerable economic concern to the livestock industry as they readily infect farmed animals such as cows (4), pigs (5), and chickens (6).In addition to the diverse range of species that they infect, coronaviruses have a propensity for host switching. For instance, HCoV-OC43 bears a strong resemblance to a bovine coronavirus, from which it probably originated (7). SARS-CoV is postulated to have originated from bats and then transferred to palm civets and finally humans (8). The MERS coronavirus probably also has its origin in bats and is responsible for severe respiratory and renal failure in humans (9). Although human-to-human transmission is low at present (10), this new beta-coronavirus has raised global health concerns because its mortality rate is more than 30% (1, 11). This "interspecies jumping" continuously threatens to initiate a novel epidemic and presents a challenge for vaccine-based containment.It is thus critical to have a better understanding of the infectious cycle of CoV. This multistep process includes attachment of the spike protein (S) to cell surface receptors, endocytosis, and then fusion of the viral and endocytic membranes (12). The viral capsid then undergoes an uncoating process to deliver the viral genome into the cytosol.Host ribosomes then translate the viral genome, yielding nonstructural proteins that modulate virus pathogenesis (13) and form with host membranes the viral transcription/replication complex (RTC). The RTC is responsible for transcription of full-length genomic RNA as well as subgenomic RNA species via a nidovirusspecific discontinuous tran...

Supramolecular Architecture of the Coronavirus Particle

“…An apparent fusion peptide of coronaviruses resides in the S2 region (Bosch et al, 2003;Sainz et al, 2005), where it is exposed by cleavage that takes place on tetraspanin-enriched membranes where host proteases including TMPRSS2 and HAT localize for some coronaviruses including MERS-CoV, while other spikes can be primed for entry by cathepsins (Bertram et al, 2013;Earnest et al, 2015;Glowacka et al, 2011;Heurich et al, 2014;Huang et al, 2006;Shulla et al, 2011;Simmons et al, 2005). This is consistent with host gene knockdown experiments that show that coronavirus entry is dependent on several elements that are important in the endosomal and lysosomal trafficking (Burkard et al, 2014;Wong et al, 2015). Expression of IFITM proteins inhibits entry driven by several coronavirus spike proteins (Huang et al, 2011;Wrensch et al, 2014), but paradoxically appears to promote infection by HCoV-OC43 (Zhao et al, 2014).…”

Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus

“…This suggests that VCP may also play a role in either the binding or entry steps of the WNV life cycle. A role for VCP in early stages of viral infection has previously been reported for coronavirus and Sindbis virus (Panda et al, 2013;Wong et al, 2015). Depletion of VCP inhibited coronavirus infection through a failure in the maturation of virus-loaded endosomes leading to accumulation of coronavirus particles in the early endosomal compartment (Wong et al, 2015).…”

“…A role for VCP in early stages of viral infection has previously been reported for coronavirus and Sindbis virus (Panda et al, 2013;Wong et al, 2015). Depletion of VCP inhibited coronavirus infection through a failure in the maturation of virus-loaded endosomes leading to accumulation of coronavirus particles in the early endosomal compartment (Wong et al, 2015). Studies on Sindbis virus indicated that VCP functioned as a regulator of viral entry as knockdown of VCP caused an alteration of trafficking and resulted in the degradation of Sindbis virus entry receptor (Panda et al, 2013).…”

“…Depletion of VCP using siRNA knockdown, resulted in accumulation of IBV particles in early endosomes as maturation of the endosome and acidification was disrupted. Failure of the acidification of virus-containing endosomes inhibited fusion between the virus envelope and endosomal membrane and prevented IBV exit from the endosomes to the cytosol (Wong et al, 2015) In the present study, we investigated whether VCP is involved in WNV infection. Specifically, we employed VCP inhibitors and siRNA knockdown to elucidate a potential role of VCP in WNV replication.…”

“…Studies of infectious bronchitis virus (IBV), family Coronaviridae, have suggested that VCP is engaged in the internalization steps of IBV (Wong et al, 2015). Depletion of VCP using siRNA knockdown, resulted in accumulation of IBV particles in early endosomes as maturation of the endosome and acidification was disrupted.…”