The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367^372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.z 1999 Federation of European Biochemical Societies.

An amino acid substitution (D --> K) in the C3 region of HIV-1 gp120 has previously been shown to inhibit binding of virions to CD4+ cells. We have introduced the same mutation into the HIV-1 isolate LAV-I(BRU), in which the mutation is denoted D373K. Here we show that the D373K envelope protein is processed and incorporated into virus particles, but that D373K virions have no detectable infectivity (below 0.1% relative to wild type). When D373K and the wild-type envelope gene were cotransfected in 293 cells at a 4:1 ratio, the resultant infectivity of the HIV-1 supernatant was reduced more than 100-fold. When the same ratio of plasmids was tested in COS-1 cells the inhibition of HIV-1 was an order of magnitude less than observed in 293 cells. COS-1 and 293 cells differed in that only 293 cells displayed saturation of virus production with respect to the envelope protein. Our data fit a simple model: when virion formation is saturated with envelope protein, expression and incorporation of a defective envelope protein imply a corresponding dilution of wild-type protein on the surface of virions. The cooperative function of wild-type envelope proteins is subsequently compromised, and a trans-dominant inhibition of virus infectivity is observed.

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.