Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication

A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with (35)S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology

Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3′ end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag

Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication

Abstract A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with 35S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology.</p

Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis▿

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D− and R+/D+) experienced a greater infection burden than the R−/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (<3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/105 polymorphonuclear leukocytes) than the R−/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection