Systematic analysis of intracellular trafficking motifs located within the cytoplasmic domain of simian immunodeficiency virus glycoprotein gp41.

Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

Diversity of envelope genes from an uncloned stock of SIVmac251.

AIDS vaccine and pathogenesis research will benefit from a more diverse array of cloned SIV challenge stocks from which to choose. Toward this end, 20 envelope genes were cloned from an extensively used, primary stock of uncloned SIVmac251. Each of the 20 clones had a unique sequence. Their translated sequences differed by as many as 26 amino acids from one another and by as many as 45 amino acids from the commonly used clone SIVmac239. Envelope sequences up to and including the membrane-spanning domain were exchanged into the infectious pathogenic SIVmac239 clone and virus stocks were produced by HEK293T cell transfection. Seventeen of the 20 recombinants were replication competent. The infectivities per ng p27 of the 17 new replication-competent recombinants in C8166-SEAP cells and in TZM-bl cells ranged from minus 32-fold to plus 7.6-fold relative to SIVmac239. A range of sensitivities to neutralization by sCD4 and by sera from SIV-infected macaques was observed but none was as sensitive to these neutralizing agents as SIVmac316, the highly macrophage-competent derivative of SIVmac239. Four strains that were most sensitive to sCD4 inhibition were also among the most sensitive to antibody-mediated neutralization. None of the new recombinant viruses replicated as well as SIVmac316 in primary alveolar macrophage cultures from rhesus monkeys but three of the strains did exhibit significant levels of delayed replication in these primary macrophages, reaching peak levels of virus production of ≥50 ng/ml p27 compared to 600-800 ng/ml p27 with SIVmac316. These new SIV clones are being contributed to the NIH AIDS Reagent Repository and are available to the scientific community.

Potent antibody-mediated neutralization and evolution of antigenic escape variants of simian immunodeficiency virus strain SIVmac239 in vivo.

Here, we describe the evolution of antigenic escape variants in a rhesus macaque that developed unusually high neutralizing antibody titers to SIVmac239. By 42 weeks postinfection, 50% neutralization of SIVmac239 was achieved with plasma dilutions of 1:1,000. Testing of purified immunoglobulin confirmed that the neutralizing activity was antibody mediated. Despite the potency of the neutralizing antibody response, the animal displayed a typical viral load profile and progressed to terminal AIDS with a normal time course. Viral envelope sequences from week 16 and week 42 plasma contained an excess of nonsynonymous substitutions, predominantly in V1 and V4, including individual sites with ratios of nonsynonymous to synonymous substitution rates (dN/dS) highly suggestive of strong positive selection. Recombinant viruses encoding envelope sequences isolated from these time points remained resistant to neutralization by all longitudinal plasma samples, revealing the failure of the animal to mount secondary responses to the escaped variants. Substitutions at two sites with significant dN/dS values, one in V1 and one in V4, were independently sufficient to confer nearly complete resistance to neutralization. Substitutions at three additional sites, one in V4 and two in gp41, conferred moderate to high levels of resistance when tested individually. All the amino acid changes leading to escape resulted from single nucleotide substitutions. The observation that antigenic escape resulted from individual, single amino acid replacements at sites well separated in current structural models of Env indicates that the virus can utilize multiple independent pathways to rapidly achieve similar levels of resistance.

A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses.

Members of the tumor necrosis factor (TNF) receptor (TNFR) superfamily are potent regulators of apoptosis, a process that is important for the maintenance of immune homeostasis. Recent evidence suggests that TNFR-1 and Fas and TRAIL receptors can also trigger an alternative form of cell death that is morphologically distinct from apoptosis. Because distinct molecular components including the serine/threonine protein kinase receptor-interacting protein (RIP) are required, we have referred to this alternative form of cell death as "programmed necrosis." We show that TNFR-2 signaling can potentiate programmed necrosis via TNFR-1. When cells were pre-stimulated through TNFR-2 prior to subsequent activation of TNFR-1, enhanced cell death and recruitment of RIP to the TNFR-1 complex were observed. However, TNF-induced programmed necrosis was normally inhibited by caspase-8 cleavage of RIP. To ascertain the physiological significance of RIP and programmed necrosis, we infected Jurkat cells with vaccinia virus (VV) and found that VV-infected cells underwent programmed necrosis in response to TNF, but deficiency of RIP rescued the infected cells from TNF-induced cytotoxicity. Moreover, TNFR-2-/- mice exhibited reduced inflammation in the liver and defective viral clearance during VV infection. Interestingly, death effector domain-containing proteins such as MC159, E8, K13, and cellular FLIP, but not the apoptosis inhibitors Bcl-xL, p35, and XIAP, potently suppressed programmed necrosis. Thus, TNF-induced programmed necrosis is facilitated by TNFR-2 signaling and caspase inhibition and may play a role in controlling viral infection.