An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity.

The HIV-1 envelope glycoprotein (Env) (gp120-gp41)3 is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4+ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.

While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

Enantioselective regulation of lactate racemization by LarR in Lactobacillus plantarum.

Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of l- and d-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the l-/d-lactate ratio and maximally induced in the presence of l-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarA induction by l-lactate. Gel retardation and footprinting experiments indicate that l-lactate has a positive effect on the binding and multimerization of LarR, while d-lactate antagonizes the positive effect of l-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.

Study of the HIV-2 Env cytoplasmic tail variability and its impact on Tat, Rev and Nef.

BACKGROUND: The HIV-2 env's 3' end encodes the cytoplasmic tail (CT) of the Env protein. This genomic region also encodes the rev, Tat and Nef protein in overlapping reading frames. We studied the variability in the CT coding region in 46 clinical specimens and in 2 reference strains by sequencing and by culturing. The aims were to analyse the variability of Env CT and the evolution of proteins expressed from overlapping coding sequences. RESULTS: A 70% reduction of the length of the CT region affected the HIV-2 ROD and EHO strains in vitro due to a premature stop codon in the env gene. In clinical samples this wasn't observed, but the CT length varied due to insertions and deletions. We noted 3 conserved and 3 variable regions in the CT. The conserved regions were those containing residues involved in Env endocytosis, the potential HIV-2 CT region implicated in the NF-kB activation and the potential end of the lentiviral lytic peptide one. The variable regions were the potential HIV-2 Kennedy region, the potential lentiviral lytic peptide two and the beginning of the potential lentiviral lytic peptide one. A very hydrophobic region was coded downstream of the premature stop codon observed in vitro, suggesting a membrane spanning region. Interestingly, the nucleotides that are responsible for the variability of the CT don't impact rev and Nef. However, in the Kennedy-like coding region variability resulted only from nucleotide changes that impacted Env and Tat together. CONCLUSION: The HIV-2 Env, Tat and Rev C-terminal part are subject to major length variations in both clinical samples and cultured strains. The HIV-2 Env CT contains variable and conserved regions. These regions don't affect the rev and Nef amino acids composition which evolves independently. In contrast, Tat co-evolves with the Env CT.