Higher-order structures in the 5 untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5 UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of ؊9.1 kcal/mol in the positive strand and ؊3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5 UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5 UTR is a cis-acting element for DI RNA replication and that its associated intra-5 UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.The 5Ј and 3Ј untranslated regions (UTR) in the coronavirus RNA genome and their counterparts in the antigenome are presumed to carry primary and higher-order structural elements important for genome replication, possibly as sites for the binding of viral RNA-dependent RNA polymerase (RdRp) and associated proteins in formation of the replication complex. For bovine coronavirus (BCoV), one molecule being used for the identification of UTR-mapping cis-acting RNA replication elements is a cloned, naturally occurring 2.13-kb defective interfering (DI) RNA that undergoes replication in the presence of BCoV helper virus (5-7). The BCoV DI RNA is composed of the fused termini of the virus genome [the 5Ј-terminal 496 nucleotides (nt) and the 3Ј-terminal 1,637 nt exclusive of the poly(A) tail, which is fused at the third nucleotide upstream of the N start codon, and a poly(A) tail of 68 nt]. To facilitate quantitation of replication by Northern assays, a 30-nt in-frame reporter sequence has been inserted at a unique BglII site at position 1093 to form construct pDrep1 (6). To date, with the use of this and DI RNA molecules from the mouse hepatitis coronavirus (MHV), another group 2 coronavirus, five putative UTR-mapping, cis-acting replication elements have been experimentally identified. Four of these show phylogenetic conservation and map within the 3Ј UTR. They include the 3Ј-terminal poly(A) tail (...

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.

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.

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An immunization regimen was evaluated in rabbits consisting of the soluble, oligomeric form of envelope glycoprotein of HIV-1, strain R2 (gp140(R2)), or the surface component of the same envelope (Env), gp120(R2), in the adjuvant AS02A. The gp140(R2) was selected based on its unusual CD4-independent phenotype and the exceptionally broad neutralizing response in the infected donor. The gp140(R2) immunogen induced antibodies that achieved 50% neutralization of 48/48, and 80% neutralization of 43/46 primary strains of diverse HIV-1 subtypes tested. The strains tested included members of standard panels of subtype B and C strains, and other diverse strains known to be neutralization resistant. The gp120(R2) induced antibodies that neutralized 9/48 of the same strains. Neutralization was IgG-mediated and HIV-1-specific. These results demonstrate that induction of truly broad spectrum neutralizing antibodies is an achievable goal in HIV-1 vaccine development.