Human immunodeficiency virus (HIV) gp41 escape mutants: cross-resistance to peptide inhibitors of HIV fusion and altered receptor activation of gp120.

Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.

Differential stability and fusion activity of Lyssavirus glycoprotein trimers.

The oligomeric structure and the fusion activity of lyssavirus glycoprotein (G) was studied by comparing G from Mokola virus (GMok) and rabies virus (PV strain) (GPV), which are highly divergent lyssaviruses. G expressed at the surface of BSR cells upon either plasmid transfection or virus infection are shown to be mainly trimeric after cross-linking experiments. However, solubilization by a detergent (CHAPS) and analysis in sucrose sedimentation gradient evidenced that GMok trimer is less stable than GPV trimer. A chimeric glycoprotein (G Mok-PV) associating the N-terminal half of GMok to the C-terminal half part of GPV formed trimers with an intermediate stability, indicating that the G C-terminal domain is essential in trimer stability. A cell to cell fusion assay revealed that GMok (and not G Mok-PV) was able to induce fusion at a higher pH (0.5 pH unit) than GPV. Such differences in the oligomeric structure stability and in the fusion activity of lyssavirus glycoproteins may partly account for the previously reported differences of their immunogenic and pathogenic properties.

Interaction of lyssaviruses with the low-affinity nerve-growth factor receptor p75NTR.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2-5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Lyssavirus glycoproteins expressing immunologically potent foreign B cell and cytotoxic T lymphocyte epitopes as prototypes for multivalent vaccines.

Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssavirus B and T cell epitopes for DNA-based immunization of mice, with the aim of developing a multivalent vaccine prototype. Truncated G (GPVIII) was composed of the C-terminal half (aa 253-503) of the Pasteur rabies virus (PV: genotype 1) G containing antigenic site III and the transmembrane and cytoplasmic domains. The chimeric G (GEBL1-PV) was composed of the N-terminal half (aa 1-250) of the European bat lyssavirus 1 (genotype 5) G containing antigenic site II linked to GPVIII. Antigenic sites II and III are involved in the induction of virus-neutralizing antibodies. The B cell epitope was the C3 neutralization epitope of the poliovirus type 1 capsid VP1 protein. The T cell epitope was the H2d MHC I-restricted epitope of the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) involved in the induction of both cytotoxic T cell (CTL) production and protection against LCMV. Truncated G carrying foreign epitopes induced weak antibody production against rabies and polio viruses and provided weak protection against LCMV. In contrast, the chimeric plasmid containing various combinations of B and CTL epitopes elicited simultaneous immunological responses against both parental lyssaviruses and poliovirus and provided good protection against LCMV. The level of humoral and cellular immune responses depended on the order of the foreign epitopes inserted. Our results demonstrate that chimeric lyssavirus glycoproteins can be used not only to broaden the spectrum of protection against lyssaviruses, but also to express foreign B and CTL epitopes. The potential usefulness of chimeric lyssavirus glycoproteins for the development of multivalent vaccines against animal diseases and zoonoses, including rabies, is discussed.