Viral entry and the ubiquitin-proteasome system.

Viruses confiscate cellular components of the ubiquitin-proteasome system (UPS) to facilitate many aspects of the infectious cycle. The 26S proteasome is an ATP-dependent, multisubunit proteolytic machine present in all eukaryotic cells. The proteasome executes the controlled degradation of functional proteins, as well as the hydrolysis of aberrantly folded polypeptides. There is growing evidence for the role of the UPS in viral entry. The UPS assists in several steps of the initiation of infection, including endosomal escape of the entering virion, intracellular transport of incoming nucleocapsids and uncoating of the viral genome. Inhibitors of proteasome activity, including MG132, epoxomicin, lactacystin and bortezomib have been integral to developments in this area. Here, we review the mechanistic details of UPS involvement in the entry process of viruses from a multitude of families. The possibility of proteasome inhibitors as therapeutic antiviral agents is highlighted.

Herpes Simplex Virus Glycoprotein C Regulates Low-pH Entry.

Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multicomponent virus-encoded machinery. Herpesviruses enter cells by endosomal low-pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via the envelope glycoproteins gB and gD and the heterodimer gH/gL regardless of pH or endocytosis requirements. Specifics concerning HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here, we demonstrate that gC regulates cell entry and infection by a low-pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold for acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low-pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes.IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and that are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry, such as entry into epithelial cells, by a low-pH pathway. gC facilitates a conformational change of the main fusogen gB, a class III fusion protein. We propose a model whereby gC functions with gB, gD, and gH/gL to allow low-pH entry. In the absence of gC, HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where gB changes occur at more acidic pHs. This report identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor.

Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action.IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib's inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.

Herpes Simplex Virus 1 Envelope Cholesterol Facilitates Membrane Fusion.

Methyl beta-cyclodextrin (MßCD) treatment of herpes simplex virus 1 (HSV-1) reduced envelope cholesterol levels and inhibited viral entry and infectivity in several cell types, regardless of the dependence of entry on endocytosis or low pH. Viral protein composition was similar in MßCD-treated and untreated virions, and ultrastructural analysis by electron microscopy revealed that cholesterol removal did not grossly affect virion structure or integrity. Removal of envelope cholesterol greatly reduced virion fusion activity as measured by fusion-from-without, suggesting that virion cholesterol is critical for the step of membrane fusion. MßCD-treatment of HSV-1 did not reduce viral attachment to the cells nor endocytic uptake of HSV-1 from the cell surface. The pre-fusion form of gB present in the HSV-1 envelope undergoes conformational changes in response to mildly acidic pH. These gB changes occurred independently of envelope cholesterol. Removal of cholesterol compromised virion stability as measured by recovery of infectivity following cycles of freeze-thaw. Taken together, the data suggest that HSV-1 envelope cholesterol is important for viral entry and infectivity due to a critical role in membrane fusion.