A G1-like state allows HIV-1 to bypass SAMHD1 restriction in macrophages.

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.

Sequential CCR5-Tropic HIV-1 Reactivation from Distinct Cellular Reservoirs following Perturbation of Elite Control.

BACKGROUND: HIV Elite Controllers may reveal insights into virus persistence given they harbour small reservoir sizes, akin to HIV non-controllers treated early with combination antiretroviral therapy. Both groups of patients represent the most promising candidates for interventions aimed at sustained remission or 'cure'. Analytic treatment interruption (ATI) in the latter group leads to stochastic rebound of virus, though it is unclear whether loss of elite control is also associated with similar rebound characteristics. METHODS: We studied three discrete periods of virus rebound during myeloma related immune disruption over 2.5 years in an elite controller who previously underwent autologous stem cell transplantation (ASCT) in the absence of any antiretroviral therapy. Single genome sequencing of the V1-V4 region of env in PBMC and plasma was performed and phylogenies reconstructed. Average pairwise distance (APD) was calculated and non-parametric methods used to assess compartmentalisation. Coreceptor usage was predicted based on genotypic algorithms. RESULTS: 122 single genome sequences were obtained (median 26 sequences per rebound). The initial rebounding plasma env sequences following ASCT represented two distinct lineages, and clustered with proviral DNA sequences isolated prior to ASCT. One of the lineages was monophyletic, possibly indicating reactivation from clonally expanded cells. The second rebound occurred 470 days after spontaneous control of the first rebound and was phylogenetically distinct from the first, confirmed by compartmentalisation analysis, with a different cellular origin rather than ongoing replication. By contrast, third rebound viruses clustered with second rebound viruses, with evidence for ongoing evolution that was associated with lymphopenia and myeloma progression. Following ASCT a shift in tropism from CXCR4-tropic viruses to a CCR5-tropic population was observed to persist through to the third rebound. CONCLUSIONS: Our data highlight similarities in the viral reservoir between elite and non-controllers undergoing ATI following allogeneic transplantation. The lack of propagation of CXCR4 using viruses following transplantation warrants further study.

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo.

Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1.

Proof-of-Principle for Immune Control of Global HIV-1 Reactivation In Vivo.

BACKGROUND: Emerging data relating to human immunodeficiency virus type 1 (HIV-1) cure suggest that vaccination to stimulate the host immune response, particularly cytotoxic cells, may be critical to clearing of reactivated HIV-1-infected cells. However, evidence for this approach in humans is lacking, and parameters required for a vaccine are unknown because opportunities to study HIV-1 reactivation are rare. METHODS: We present observations from a HIV-1 elite controller, not treated with combination antiretroviral therapy, who experienced viral reactivation following treatment for myeloma with melphalan and autologous stem cell transplantation. Mathematical modeling was performed using a standard viral dynamic model. Enzyme-linked immunospot, intracellular cytokine staining, and tetramer staining were performed on peripheral blood mononuclear cells; in vitro CD8 T-cell-mediated control of virion production by autologous CD4 T cells was quantified; and neutralizing antibody titers were measured. RESULTS: Viral rebound was measured at 28,000 copies/mL on day 13 post-transplant before rapid decay to <50 copies/mL in 2 distinct phases with t1/2 of 0.71 days and 4.1 days. These kinetics were consistent with an expansion of cytotoxic effector cells and killing of productively infected CD4 T cells. Following transplantation, innate immune cells, including natural killer cells, recovered with virus rebound. However, most striking was the expansion of highly functional HIV-1-specific cytotoxic CD8 T cells, at numbers consistent with those applied in modeling, as virus control was regained. CONCLUSIONS: These observations provide evidence that the human immune response is capable of controlling coordinated global HIV-1 reactivation, remarkably with potency equivalent to combination antiretroviral therapy. These data will inform design of vaccines for use in HIV-1 curative interventions.

Vpx complementation of 'non-macrophage tropic' R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages.

BACKGROUND: It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses. RESULTS: Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS). CONCLUSIONS: Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for 'non-macrophage tropic' viruses.

Macrophages: the neglected barrier to eradication.

PURPOSE OF REVIEW: There has been a shift towards HIV-1 eradication research in the last three years, yet much is still unknown about the precise role that macrophages will play in any such strategy. This review attempts to summarize the latest data on this subject. RECENT FINDINGS: A new generation of histone deacetylase inhibitors, ITF2357, belinostat, givinostat, panobinostat, and the cancer drug JQ1, have been shown to induce viral reactivation in a monocyte cell line. In macrophages chronically infected with HIV-1 in vitro, drugs blocking pre-integration steps have demonstrated poor efficacy in controlling viral replication in comparison to protease inhibitors, thus questioning whether drugs can control this reservoir following histone deacetylase inhibition. Finally, non-human primate data suggest that CD8+ T cells may not be able to clear infected macrophages. SUMMARY: Given these data highlighting the barriers to addressing the macrophage reservoir, functional rather than sterilizing cure may be a realistic goal. More research on macrophages is needed and animal models may prove useful in future HIV-1 eradication studies by offering a clinically relevant way to study macrophage infection in vivo.