Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.The Env glycoprotein complex mediates receptor binding and membrane fusion during human immunodeficiency virus type 1 (HIV-1) infection of susceptible cells (66). It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid). The gp120 and gp41 proteins then remain noncovalently associated, forming the functional, native gp120 3 -gp41 3 complex (20,24,66).During fusion, the gp120 protein interacts with the virus receptor and coreceptor on target cells. This triggers conformational changes that lead to the insertion of a hydrophobic fusion peptide, located at the N terminus of gp41, into the target cell membrane (66). Cleavage of gp160 is essential for fusion, since uncleaved gp160 is fusion incompetent (9,33,39,48). Generally, only cleaved Env is incorporated into virions (22), although uncleaved Env can be virion associated (39,48). By analogy with other enveloped viruses such as influenza A virus (5,...

A reliable method for the quantitation of plasma viremia in nonhuman primates infected with simian immunodeficiency virus (SIV) and related viruses is described. This method is based on an established quantitative-competitive PCR format and includes a truncated control for internal assay calibration. Optimization of assay conditions has significantly improved amplification specificity, and interassay variability is comparable to that of commercially available assays for human immunodeficiency virus (HIV) quantitation. This procedure was used to monitor viral loads in a group of Macaca mulatta animals that were infected with SIVsmE660 for over 2 years. Highly diverse profiles of plasma viremia were observed among animals, and high viral loads were associated with more rapid disease progression. Spearman rank correlation analyses were done for survival versus three parameters of viral load: plasma viremia, p27 core antigen, and frequency of infected peripheral blood mononuclear cells. Plasma viremia had the strongest overall correlation and was significantly (P < 0.05 to P < 0.01) associated with survival at 10 of the 13 time points examined. Plasma viremia did not correlate with survival during the primary viremia phase; however, the strength of this correlation increased with time postinfection and, remarkably, viremia levels as early as week 6 postinfection were highly predictive (P < 0.01) of relative survival. These findings are consistent with the available clinical data concerning viral load correlates early in HIV infection, and they provide further support for the view that disease outcome in lentiviral infection may be largely determined by events that occur shortly after infection.

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We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gpl20 and gp4l. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gpl20-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development. * Corresponding author. ing of gpl60 in mammalian cells (16a). The HIV p55 protein is anchored in cellular membranes by a myristic acid moiety added to the N-terminal penultimate glycine residue (2). Similar to other retrovirus systems (33), anchoring of the HIV-1 core protein has been shown to be necessary for production of virions (14). Development of an in vitro recombinant expression system that mimics the HIV-1 virus synthesis and maturation pathways would be advantageous for studying the role of env and gag protein association in regulating virus formation and release. We (S.

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Anti-human immunodeficiency virus type 1 (Anti-HIV-1) antibody response was compared in four groups of mice following inoculation with HIV-1 gp160, with live recombinant vaccinia virus expressing HIV-1 envelope glycoproteins, or with both immunogens in alternate orders for primary or secondary immunizations. Both subunit and recombinant virus immunogens induced similar levels of antibody response following primary immunization. However, after secondary immunization, mice primed with live recombinant virus and then boosted with subunit gp160 immunogen showed significantly higher antibody response than those in the other three groups. Neutralizing antibodies were generated only in this group of mice and were shown to neutralize both the homologous virus (BRU) and a divergent isolate (SF2) of HIV-1. On the other hand, their reactivities to peptide sequences from the principal neutralizing determinant (PND) of gp120 were limited to the BRU isolate, not SF2 or MN, indicating that the cross-neutralizing activities were directed against determinants other than the linear epitope(s) within the PND. These results also indicate that combined immunization by priming with liver recombinant virus and boosting with subunit immunogen may be more effective than immunization by either immunogen alone.

Simian AIDS (SAIDS) is an endemic disease of macaques that shares many characteristics with AIDS in humans. SAIDS is etiologically linked to infection by a type D retrovirus, SAIDS retrovirus (SRV). Immunization with an inactivated whole-virus vaccine was shown to protect macaques against infection by SRV serotype 1. To identify the antigen(s) responsible for eliciting protective immunity, we have constructed a recombinant vaccinia virus (v-senv5) that expresses the envelope glycoproteins of SRV serotype 2 (SRV-2/W). Pig-tailed macaques (Macaca nemestrina) immunized with vsenv5 showed lymphoproliferative responses to purified SRV-2/W. They also generated antibodies that neutralized SRV-2/W infectivity in vitro and mediated antibody-dependent cellular cytotoxicity against SRV-2-infected cells. Four v-senv5-immunized animals, together with four control animals, were challenged intravenously with 5 X 103 tissue culture infectious doses of SRV-2/W. As early as 2 weeks after challenge, three of four control animals became viremic, and two of these three animals also seroconverted. The animal that was viremic but remained antibody negative died of symptoms of SRV infection 6/2 weeks after challenge. In contrast, all four v-senv5-immunized animals remained healthy, virus-free, and seropositive against only the immunizing envelope antigens. These results indicate that immunization with a recombinant vaccinia virus expressing the envelope antigens of SRV-2/W protects primates from infection by a retrovirus that causes immunodeficiency diseases.