Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Background

Dendritic cells (DCs) are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS) induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN) partially inhibits HIV-1 replication and cell-cell transmission in CD4⁺ T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4⁺ T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown.

Results

We demonstrated that IFN-alpha (IFNα)-induced mature DCs restricted HIV-1 replication and trans-infection of CD4⁺ T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4⁺ T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner.

Conclusions

The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.     

Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: one step closer to personalized medicine?

Background

It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication of a lentivirus such as HIV-1 after the deliberate intragenomic insertion of a bona fide miRNA.

Results

We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication.

Conclusion

This is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.     

The microRNA miR-29a is associated with human immunodeficiency virus latency

Background

Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3′LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency.

Findings

We used two established cellular models of HIV-1 latency – the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication.

Conclusions

The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

     

Stable HIV-1 integrase diversity during initial HIV-1 RNA Decay suggests complete blockade of plasma HIV-1 replication by effective raltegravir-containing salvage therapy

Background

There is legitimate concern that minority drug-resistant mutants may be selected during the initial HIV-1 RNA decay phase following antiretroviral therapy initiation, thus undermining efficacy of treatment. The goal of this study was to characterize viral resistance emergence and address viral population evolution during the first phase of viral decay after treatment containing initiation.

Findings

454 sequencing was used to characterize viral genetic diversity and polymorphism composition of the HIV-1 integrase gene during the first two weeks following initiation of raltegravir-containing HAART in four ART-experienced subjects. No low-prevalence Raltegravir (RAL) drug resistance mutations (DRM) were found at baseline. All patients undergoing treatment received a fully active ART according to GSS values (GSS?≥?3.5). No emergence of DRM after treatment initiation was detected. Longitudinal analysis showed no evidence of any other polymorphic mutation emergence or variation in viral diversity indexes.

Conclusions

This suggests that fully active salvage antiretroviral therapy including raltegravir achieves a complete blockade of HIV-1 replication in plasma. It is unlikely that raltegravir-resistant HIV-1 may be selected in plasma during the early HIV-1 RNA decay after treatment initiation if the administered therapy is active enough.

     

Genome-wide association study identifies single nucleotide polymorphism in DYRK1A associated with replication of HIV-1 in monocyte-derived macrophages

Background

HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages.

Methodology/Principal Findings

Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16×10⁻⁵). While the association was not genome-wide significant (p<1×10⁻⁷), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84×10⁻⁶). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048).

Conclusions/Significance

These findings suggest that the kinase DYRK1A is involved in the replication of HIV-1, in vitro in macrophages as well as in vivo.     

HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk

Background

HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs.

Methodology/Principal Findings

Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1_BaL or X4-HIV-1_NDK, and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.

Conclusion

These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1 in the triggering of HIV-1 replication and replenishment of viral reservoirs in AIDS.     

Lentiviral Nef suppresses iron uptake in a strain specific manner through inhibition of Transferrin endocytosis

Background

Increased cellular iron levels are associated with high mortality in HIV-1 infection. Moreover iron is an important cofactor for viral replication, raising the question whether highly divergent lentiviruses actively modulate iron homeostasis. Here, we evaluated the effect on cellular iron uptake upon expression of the accessory protein Nef from different lentiviral strains.

Results

Surface Transferrin receptor (TfR) levels are unaffected by Nef proteins of HIV-1 and its simian precursors but elevated in cells expressing Nefs from most other primate lentiviruses due to reduced TfR internalization. The SIV Nef-mediated reduction of TfR endocytosis is dependent on an N-terminal AP2 binding motif that is not required for downmodulation of CD4, CD28, CD3 or MHCI. Importantly, SIV Nef-induced inhibition of TfR endocytosis leads to the reduction of Transferrin uptake and intracellular iron concentration and is accompanied by attenuated lentiviral replication in macrophages.

Conclusion

Inhibition of Transferrin and thereby iron uptake by SIV Nef might limit viral replication in myeloid cells. Furthermore, this new SIV Nef function could represent a virus-host adaptation that evolved in natural SIV-infected monkeys.     

Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies

Background

An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300.

Results

In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process.

Conclusions

The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.     

Early and prolonged antiretroviral therapy is associated with an HIV-1-specific T-cell profile comparable to that of long-term non-progressors

Background

Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).

Methodology/Principal Findings

We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4⁺ and CD8⁺ T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-γ, IL-2, TNF-α production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4⁺ and CD8⁺ T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8⁺ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.

Conclusions

Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.     

Dendritic cells reveal a broad range of MHC class I epitopes for HIV-1 in persons with suppressed viral load on antiretroviral therapy

Background

HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8⁺ T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8⁺ T cells, and could potentially be targeted to activate memory CD8⁺ T cells to a broad array of HIV-1 epitopes during ART.

Principal Findings

We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8⁺ T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a.

Significance

There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8⁺ T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.     

HIV-1 Activates Macrophages Independent of Toll-Like Receptors

Background

Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1). Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection.

Methodology/Principal Findings

To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK), and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR) pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1β, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication.

Conclusions/Significance

HIV-1 induced a primed, proinflammatory state, M1_HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic approaches.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads

Background

HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression.

Methodology/Principal Findings

We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individual''s HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads.

Conclusions/Significance

This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.     

Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection

Background

HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.

Methodology/Principal Findings

We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.

Conclusions/Significance

Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.     

Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background

HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus.

Methodology and Principal Findings

Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides.

Conclusion and Significance

This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.