X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven alpha-helices, five of which are arranged in a coiled coil-like structure. Sequence assignments were made for two of the helices, and tentative connectivity of the remainder of the protein was confirmed by the recent solution structure of a monomeric N-terminal fragment. The C-terminal third of the protein is mostly disordered in the crystal. The longest helices in the coiled coil-like structure are separated by a long, highly antigenic peptide that includes the binding site of an antibody fragment complexed with CA in the crystal. The site of binding of the Fab, the position of the antigenic loop and the site of cleavage between the matrix protein and CA establish the side of the dimer that would be on the exterior of the retroviral core.

Highlights d HIV cure studies rely on precise HIV reservoir assays that can be done at scale d Multiplexed droplet digital PCR can probe multiple HIV genome targets per sample d HIV-1 proviruses containing 5 probed genomic regions are likely intact d PCR-based counting of T cells improves reservoir quantification in tissues

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Monoclonal antibodies specific for the gpl20 or gp4l portions of the human immunodeficiency virus (HIV) envelope protein gpl60 were conjugated to ricin A chain, and their immunotoxic activities against HIV-infected cells were evaluated in the presence or absence of soluble CD4 (sCD4).Immunotoxin activity was measured in vitro as cytotoxicity and inhibition of secretion of infectious HIV. The efficacy of anti-gp4l immunotoxins was enhanced at least 30-fold in the presence of sCD4. This effect was specific for HIV-infected cells, but not for uninfected cells, and was seen at concentrations of sCD4 as low as 0.1 pg/mI. Anti-gpl20 immunotoxins were marginally inhibited at higher concentrations of sCD4. Flow cytometry analyses showed that sCD4 increased the expression of gp4l on the surface of infected cells and increased internalization of gpl20 and gp4l. These data suggest that sCD4 alters the cellular trafficking of HIV envelope proteins. These findings also have important implications for the therapeutic use of anti-HIV immunotoxins and may be generalizable to other immunotoxins as well.

BackgroundHIV-1 is frequently detected in the lungs of infected individuals and is likely important in the development of pulmonary opportunistic infections. The unique environment of the lung, rich in alveolar macrophages and with specialized local immune responses, may contribute to differential evolution or selection of HIV-1.Methodology and FindingsWe characterized HIV-1 in the lung in relation to contemporaneous viral populations in the blood. The C2-V5 region of HIV-1 env was sequenced from paired lung (induced sputum or bronchoalveolar lavage) and blood (plasma RNA and proviral DNA from sorted or unsorted PBMC) from 18 subjects. Compartmentalization between tissue pairs was assessed using 5 established tree or distance-based methods, including permutation tests to determine statistical significance. We found statistical evidence of compartmentalization between lung and blood in 10/18 subjects, although lung and blood sequences were intermingled on phylogenetic trees in all subjects. The subject showing the greatest compartmentalization contained many nearly identical sequences in BAL sample, suggesting clonal expansion may contribute to reduced viral diversity in the lung in some cases. However, HIV-1 sequences in lung were not more homogeneous overall, nor were we able to find a lung-specific genotype associated with macrophage tropism in V3. In all four subjects in whom predicted X4 genotypes were found in blood, predicted X4 genotypes were also found in lung.ConclusionsOur results support a picture of continuous migration of HIV-1 between circulating blood and lung tissue, with perhaps a very limited degree of localized evolution or clonal replication.