Blocking HSV-1 glycoprotein K binding to signal peptide peptidase reduces virus infectivity in vitro and in vivo

HSV glycoprotein K (gK) is an essential herpes protein that contributes to enhancement of eye disease. We previously reported that gK binds to signal peptide peptidase (SPP) and that depletion of SPP reduces HSV-1 infectivity in vivo. To determine the therapeutic potential of blocking gK binding to SPP on virus infectivity and pathogenicity, we mapped the gK binding site for SPP to a 15mer peptide within the amino-terminus of gK. This 15mer peptide reduced infectivity of three different virus strains in vitro as determined by plaque assay, FACS, and RT-PCR. Similarly, the 15mer peptide reduced ocular virus replication in both BALB/c and C57BL/6 mice and also reduced levels of latency and exhaustion markers in infected mice when compared with control treated mice. Addition of the gK-15mer peptide also increased the survival of infected mice when compared with control mice. These results suggest that blocking gK binding to SPP using gK peptide may have therapeutic potential in treating HSV-1-associated infection.

Human Endogenous Retrovirus K(HML-2) Gag- and Env-Specific T-Cell Responses Are Infrequently Detected in HIV-1-Infected Subjects Using Standard Peptide Matrix-Based Screening

ABSTRACTT-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping.

Prochemerin Is a New Substrate for Thrombin.

Abstract 3591 Poster Board III-528 Introduction Prochemerin is a 163 amino acid precursor protein with a C-terminal domain highly susceptible to proteolysis. Its chemotactic activity is unmasked upon C-terminal cleavage by proteases of the coagulation, fibrinolytic and inflammatory systems. Here, we studied whether thrombin is able to cleave prochemerin to generate active forms of chemerin. Methods The 15mer peptide prochemerin C-terminal sequence (YFPGQFAFSKALPRS) or prochemerin was incubated with thrombin at different concentrations and times. The reactions were stopped by addition of PPACK before determining their chemotactic activity in a transwell assay using CMKLR1-transfected cells and their mass by mass-spectrometry. Results Thrombin (0-100 nM) dose-dependently cleaved 15mer to 14mer (YFPGQFAFSKALPR). Over a longer reaction time, the 10mer (YFPGQFAFSK) was also detected. The 15mer was almost inert in the chemotaxis assay but thrombin-cleaved 15mer caused migration of CMKLR1 transfectants. The 14mer and 10mer at 1 μM induced CMKLR1 cell migration at a rate of 3200 and 2800 cells/ml, but 1 μM 15mer did not induce any chemotaxis. Thrombin can also cleave the 14mer to a 10mer as determined by mass spectrometry. Using selected thrombin mutants for the Na+ binding site (E229K) and the active site YPPW-insertion loop (W50A), we found that thrombin hydrolysis of the 15mer was dependent on the Na+ bound “fast” form of thrombin and active site topology. The 10mer could be further activated by carboxypeptidase N (CPN) by removing the C-terminal lysine, whereas the C-terminal arginine of 14mer could not be cleaved by CPN, which did not affect its activity. Full-length prochemerin was also activated by thrombin (100nM) and the chemotactic activity further increased by CPN (50nM) about 6 fold. Conclusions Prochemerin is a new substrate for thrombin. Thrombin-cleaved chemerins are active chemoattractants in chemotaxis. This extends the molecular link between blood coagulation and CMKLR1-mediated immune responses. Disclosures: No relevant conflicts of interest to declare.