Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) in
conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons in
V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142
glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-
gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.

HIV-1 Infection of T cells and macrophages are differentially modulated by virion-associated Hck: a nef-dependent phenomenon

The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell.

Visualising single molecules of HIV-1 and miRNA nucleic acids

Background
The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.

Results
We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.

Conclusions
Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Impact of HIV-associated conditions on mortality in people commencing anti-retroviral therapy in resource limited settings

Objectives: To identify associations between specific WHO stage 3 and 4 conditions diagnosed after ART initiation and all cause mortality for patients in resource-limited settings (RLS).

Design, Setting: Analysis of routine program data collected prospectively from 25 programs in eight countries between 2002 and 2010.

Subjects, Participants: 36,664 study participants with median ART follow-up of 1.26 years (IQR 0.55–2.27).

Outcome Measures: Using a proportional hazards model we identified factors associated with mortality, including the occurrence of specific WHO clinical stage 3 and 4 conditions during the 6-months following ART initiation.

Results: There were 2922 deaths during follow-up (8.0%). The crude mortality rate was 5.41 deaths per 100 person-years (95% CI: 5.21–5.61). The diagnosis of any WHO stage 3 or 4 condition during the first 6 months of ART was associated with
increased mortality (HR: 2.21; 95% CI: 1.97–2.47). After adjustment for age, sex, region and pre-ART CD4 count, a diagnosis of extrapulmonary cryptococcosis (aHR: 3.54; 95% CI: 2.74–4.56), HIV wasting syndrome (aHR: 2.92; 95%CI: 2.21 -3.85), nontuberculous mycobacterial infection (aHR: 2.43; 95% CI: 1.80–3.28) and Pneumocystis pneumonia (aHR: 2.17; 95% CI 1.80–3.28) were associated with the greatest increased mortality. Cerebral toxoplasmosis, pulmonary and extra-pulmonary
tuberculosis, Kaposi’s sarcoma and oral and oesophageal candidiasis were associated with increased mortality, though at lower rates.

Conclusions: A diagnosis of certain WHO stage 3 and 4 conditions is associated with an increased risk of mortality in those initiating ART in RLS. This information will assist initiatives to reduce excess mortality, including prioritization of resources for
diagnostics, therapeutic interventions and research.

A novel approach to priority-setting for HIV prevention among adults in Uganda

 The study used economic and ethical theory, empirical evidence and local stakeholders views to develop a checklist of features of an ideal approach to priority-setting. This was used to determine the ideal approach for priority-setting for HIV prevention in Uganda. The Assessing Cost-Effectiveness (ACE) approach was determined to be the ideal approach using the checklist developed. A pilot study using the approach determined that in Uganda, blood safety and HIV counselling interventions are the most cost-effective, equitable and acceptable interventions. Mass media interventions were the least cost-effective and affordable, had a weak evidence base but were more acceptable. On the other hand interventions targeting Most-at-risk-populations were cost-effective but least acceptable. The study demonstrates the importance of balancing technical rigor and due process to ensure buy-in by stakeholders.

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA.

Analysis of serious non-AIDS events among HIV-infected adults at Latin American sites

Acquired immune deficiency appears to be associated with serious non-AIDS (SNA)-defining conditions such as cardiovascular disease, liver and renal insufficiency and non-AIDS-related malignancies. We analysed the incidence of, and factors associated with, several SNA events in the LATINA retrospective cohort.

Sex differences in the Toll-like receptor-mediated response of plasmacytoid dendritic cells to HIV-1

Manifestations of viral infections can differ between women and men, and marked sex differences have been described in the course of HIV-1 disease. HIV-1-infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon-alpha (IFN-alpha) in response to HIV-1-encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8(+) T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8(+) T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1-associated pathology.

Fifteen to twenty percent of HIV substitution mutations are associated with recombination

HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From >7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10(-5) mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.