The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.