Hantaanvirus (HTNV) is the prototype of the genus Hantavirus, which belongs to the family Bunyaviridae. Hantaviruses are carried and transmitted by rodents and are known to cause two serious disease syndromes in humans i.e., hemorrhagic fever with renal syndrome (HFRS) and the hantavirus pulmonary syndrome (HPS). HTNV is an enveloped virus that contains a tripartite genome consisting of three negative-sense RNA segments (L, M, S), and the S and M segment of HTNV, respectively, encode the viral nucleocapsid protein (NP) and envelope glycoproteins. Possible phosphorylation motifs of casein kinase II (CKII) and protein kinase C (PKC) were identified in HTNV NP through bioinformatics searches. Sucrose gradient SDS-PAGE analysis indicated that dephosphorylated HTNV NP migrated faster than non-dephosphorylated NP, suggesting that HTNV NP is phosphorylated in infected Vero E6 cells. Immunoblot anaylsis of HTNV particles with anti-phosphoserine antibody and anti-phosphothreonine antibody after immunoprecipitation showed that viral particles are readily phosphorylated at threonine residues. In vitro kinase assay further showed that HTNV NP is phosphorylated by CK II, but not by PKC. Full length or truncated HTNV NPs expressed in E. coli were phosphorylated in vitro by CKII suggesting that phosphorylation may occur in vivo at multiple sites. Site specific mutagenesis studies suggest that HTNV NP phosphorylation might occur at unknown sites excluding the site-directly mutagenized locations. Taken together, HTNV NP can be phosphorylated mainly at threonine residues in vivo by CK II treatment.

Background: The precise mechanism by which CTLA-4 regulates T cell immune responses is still not fully understood. Previously we proposed that CTLA-4 could downregulate T cell function by modulating a signaling cascade initiated from the T cell receptor complex. The evidence for this notion comes from our findings that CTLA-4 associated with the T cell receptor zeta (TCR zeta) chain, and hence regulated TCR zeta phosphorylation by co-associated SHP-2 tyrosine phosphatase (1). In this report, we investigated whether any other signaling molecules could be involved in the CTLA-4 signaling pathway. Methods: We have taken biochemical approaches, such as immunoprecipitation followed by autoradiography or immunoblotting, to identify the molecules associated with CTLA-4. To perform these assays, we used activated primary T cells and ectopically transfected 293 cells. Various truncation mutants of CTLA-4 were used to map the interaction site on CTLA-4. Results: We found that in addition to TCR zeta and SHP-2, a recently cloned small adaptor molecule, SAP (SLAM-associated protein), was also able to associate with CTLA-4. We identified the domain of SAP association in CTLA-4 being a motif involving GVYVKM. This motif has been previously found to bind SHP-2 through its phosphorylated tyrosine interaction with SH-2 domain of SHP-2. Indeed, co-expression of SAP and SHP-2 reduced their binding to CTLA-4 significantly, suggesting that SAP and SHP-2 compete for the common binding site, GVYVKM. Thus, by blocking SHP-2 recruitment SAP could function as a negative regulator of CTLA-4. Conclusion: Taken together, our data suggest the existence of complicate signaling cascade in regulating CTLA-4 function, and further provide evidence that SAP can act either as a positive or negative regulator depending on the nature of the associating receptors.