Bypass suppression of small-plaque phenotypes by a mutation in poliovirus 2A that enhances apoptosis.

The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin(+) (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin(-) and Sin(+) phenotypes in animal models, Sin(-) mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin(-) mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin(-) mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin(-) mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin(-) phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin(-) mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.

The D0 domain of KIR3D acts as a major histocompatibility complex class I binding enhancer.

In contrast to the KIR2D:HLA-C interaction, little is known of KIR3DL1's interaction with HLA-B or the role of D0, the domain not present in KIR2D. Differences in the strength and specificity for major histocompatibility complex class I of KIR3DL1 and its common chimpanzee homologue Pt-KIR3DL1/2 were exploited to address these questions. Domain-swap, deletion, and site-directed mutants of KIR3DL1 were analyzed for HLA-B binding using a novel, positively signaling cell-cell binding assay. Natural 'deletion' of residues 50 and 51 from its D0 domain causes Pt-KIR3DL1/2 to bind Bw4(+) HLA-B allotypes more avidly than does KIR3DL1. Deletion of these residues from KIR3DL1, or their substitution for alanine, enhanced binding of Bw4(+) HLA-B. None of 15 different point mutations in D0 abrogated KIR3DL1 binding to Bw4(+) HLA-B. In contrast point mutations in the D1 and D2 domains of KIR3DL1, made from knowledge of KIR2D:HLA-C interactions, disrupted binding to Bw4(+) HLA-B. The results are consistent with a model in which D1 and D2 make the principal contacts between KIR3DL1 and HLA-B while D0 acts through a different mechanism to enhance the interaction. This modulatory role for D0 is compatible with natural loss of expression of the D0 domain, a repeated event in the evolution of functional KIR genes.