4 research outputs found
Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide▿
Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae
Identification and Characterization of a New Chemotype of Noncovalent SENP Inhibitors
Enzymes called SENPs catalyze both
the maturation of small ubiquitin-like
modifier (SUMO) precursors and removal of SUMO modifications, which
regulate essential cellular functions such as cell cycle progression,
DNA damage response, and intracellular trafficking. Some members,
such as SENP1, are potential targets for developing cancer therapeutics.
We searched for small molecule inhibitors of SENPs using <i>in
silico</i> screening in conjunction with biochemical assays and
identified a new chemotype of small molecule inhibitors that noncovalently
inhibit SENPs. The inhibitors confer the noncompetitive inhibitory
mechanism, as shown by nuclear magnetic resonance (NMR) and quantitative
enzyme kinetic analysis. The NMR data also provided evidence for substrate-assisted
inhibitor binding, which indicates the need for caution in using artificial
substrates for compound screening, as the inhibitory effects could
be significantly different from using the physiological substrates.
This finding also suggests the possibility of designing inhibitors
for this class of enzymes that are tuned for substrate-specificity
A Potent Lassa Virus Antiviral Targets an Arenavirus Virulence Determinant
Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junı´n virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virusyield reduction assay, we determined that LHF-535 potently inhibited Junı´n virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535- treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drugresistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses