؊ protein synthesis defect, and the rescue by secondary mutations in vhs, occurred at the mRNA and/or translational levels, quantitative reverse transcriptase PCR (qRT-PCR) and polysome analyses were performed. We found that the absence of VP22 caused a small decrease in mRNA levels as well as a defect in polysome assembly that was independent of mRNA abundance. Both defects were complemented by the secondary mutations in vhs, indicating functional interplay between VP22 and vhs in both accumulation and translation of viral mRNAs.

A developmentally regulated, 160-kDa trypomastigote surface glycoprotein was previously shown to bind the third component of complement and to inhibit activation of the alternative complement pathway, thus providing the parasites a means of avoiding the lytic effects of complement. We now show that this complement regulatory protein (CRP) binds human C4b, a component of the classical pathway C3 convertase, and may therefore also act to restrict classical complement activation. Characterization of the extent of carbohydrate modification of the protein revealed extensive N-linked glycosylation and no apparent O-linked sugars. The CRP purified from parasites treated with an inhibitor of N-linked glycosylation exhibited a decreased binding affinity for C3b compared with that of the fully glycosylated protein. We have previously shown that the protein was anchored to the membrane via a glycosyl phosphatidylinositol linkage and was spontaneously shed from the parasite surface. The spontaneous release of CRP from the parasite surface may augment the protection of the parasites from complement-mediated lysis by the removal of complement-CRP complexes. The majority of the shed CRP had an apparent molecular mass of 160 kDa and lacked the glycolipid anchor, whereas the membrane form was recovered with the glycolipid anchor attached and had an apparent molecular mass of 185 kDa. Both the membrane form (185 kDa) and the soluble form (160 kDa) retained binding affinity for C3b. Evidence is presented to indicate that the conversion of the 185-kDa membrane form to the 160-kDa form is the result of cleavage by an endogenous phospholipase C.

Trypanosoma cruzi trypomastigotes are exquisitely resistant to the lytic effects of vertebrate complement, and this characteristic contributes to the survival of the parasites in the host bloodstream. Trypomastigotes avoid complement-mediated lysis by the production of a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase, thus preventing activation and amplification of the complement cascade at the parasite surface. We have developed a monoclonal antibody to the 160-kDa T. cruzi complement regulatory protein (CRP) and describe a one-step immunoaffinity purification procedure. The CRP was purified to homogeneity and subjected to amino-terminal peptide sequence analysis. Based on the protein sequence obtained, the CRP was identified as a member of a large family of trypomastigote-specific genes, and a complete cDNA was isolated and sequenced. The complete coding sequence was cloned in Escherichia coli, and antibodies raised against the full-length recombinant protein reacted specifically with a 160-kDa protein in trypomastigote membrane protein preparations as well as with native, purified CRP. Indirect immunofluorescence revealed that the protein is uniformly expressed at the cell surfaces of trypomastigotes.

Herpes simplex virus 2 (HSV-2) strains containing mutations in the virion host shutoff (vhs) protein are attenuated for replication compared with wild-type virus in mouse embryonic fibroblasts (MEFs). However, HSV-2 vhs mutants replicate to near wild-type levels in the absence of the RNA-activated protein kinase (PKR). PKR is one of several kinases that phosphorylates the eukaryotic initiation factor 2␣ (eIF2␣) to inhibit translation initiation, and we previously found that more of the phosphorylated form of eIF2␣ accumulates in MEFs infected with HSV-2 vhs mutants than with wild-type virus. Here, we show that this increase in phosphorylated eIF2␣ is primarily PKR dependent. Using MEFs expressing nonphosphorylatable eIF2␣, we demonstrate that phosphorylated eIF2␣ is the primary cause of attenuated replication of HSV-2 vhs mutants and that attenuation correlates with decreased accumulation of viral proteins. Normally, HSV antagonizes eIF2␣ phosphorylation through the action of ICP34.5, which redirects protein phosphatase 1␣ (PP1␣) to dephosphorylate eIF2␣ during infection. We show that ICP34.5 does not accumulate efficiently in MEFs infected with HSV-2 vhs mutant viruses, suggesting that the accumulation of phosphorylated eIF2␣ and the attenuated phenotype of HSV-2 vhs mutants in MEFs result from a deficiency in ICP34.5.

The generation of nitric oxide (NO) is largely responsible for the intracellular killing of Trypanosoma cruzi by activated macrophages. The present study was carried out to determine whether the production of NO by activated murine macrophages cultured in physiologic levels of arginine can be augmented by increasing the availability of arginine, the substrate for NO biosynthesis. Increased exogenous arginine or citrulline resulted in a significant increase in NO production and complete clearance of the parasites by activated macrophages. As citrulline fully substituted for arginine in supporting NO production and trypanocidal activity, these results demonstrate the expression of a highly active citrulline-NO cycle in activated macrophages and that levels of arginine in plasma are limiting with respect to both NO production and trypanocidal activity in these cells. The results indicate that increasing plasma substrate levels for both arginine and NO biosynthesis may represent a means of enhancing microbicidal activity in vivo.