Processing Of Glycoprotein Precursor Research Articles

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human cells is the processing of the viral envelope glycoprotein by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). In order to break the species barrier during zoonotic transmission and cause severe disease in humans, newly emerging arenaviruses must be able to hijack human SKI-1/S1P efficiently. Here we implement a newly developed cell-based molecular sensor for human SKI-1/S1P to characterize the processing of arenavirus glycoproteins in a quantitative manner. We further use our sensor to correctly predict efficient processing of the glycoprotein of the newly emergent pathogenic Lujo virus by human SKI-1/S1P. Our sensor thus represents a rapid and robust test system with which to assess whether the glycoprotein of any newly emerging arenavirus can be efficiently processed by human SKI-1/S1P, based solely on sequence information.

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.

Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

Targeting the Proteolytic Processing of the Viral Glycoprotein Precursor Is a Promising Novel Antiviral Strategy against Arenaviruses

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

Arenavirus Z-Glycoprotein Association Requires Z Myristoylation but Not Functional RING or Late Domains

Generation of infectious arenavirus-like particles requires the virus RING finger Z protein and surface glycoprotein precursor (GPC) and the correct processing of GPC into GP1, GP2, and a stable signal peptide (SSP). Z is the driving force of arenavirus budding, whereas the GP complex (GPc), consisting of hetero-oligomers of SSP, GP1, and GP2, forms the viral envelope spikes that mediate receptor recognition and cell entry. Based on the roles played by Z and GP in the arenavirus life cycle, we hypothesized that Z and the GPc should interact in a manner required for virion formation. Here, using confocal microscopy and coimmunoprecipitation assays, we provide evidence for subcellular colocalization and biochemical interaction, respectively, of Z and the GPc. Our results from mutation-function analysis reveal that Z myristoylation, but not the Z late (L) or RING domain, is required for Z-GPc interaction. Moreover, Z interacted directly with SSP in the absence of other components of the GPc. We obtained similar results with Z and GPC from the prototypical arenavirus lymphocytic choriomeningitis virus and the hemorrhagic fever arenavirus Lassa fever virus.