Mapping HIV-1's evasion of host defense

The HIV-1 RNA genome encodes the main structural polyprotein Gag, Gag/Pol (structural proteins and enzymes), auxiliary (Vpr, Vpu, Vif) and regulatory proteins (Tat, Rev, Nef). The expression of the full complement of viral gene products enhances both the replication and the pathogenic potential of the virus. In our previous work we showed that HIV-1 expression prevented eIF2alpha-dependent stress granule (SG) assembly in cells. SGs are translationally silent sites of RNA triage and can be readily visualized in cells due to clustering of cytoplasmic RNA and the recruitment of multiple cytoplasmic proteins (e.g., TIA/R, G3BP and eIF3). In this work, we attempted to understand how HIV-1 blocks SG formation and defined the determinant in HIV-1 that mediates this. To initiate SG assembly, the drug Pateamine A was used. Pateamine A causes an activation of eIF4A, which will lead to a disassembly of the eIF4F complex and an arrest in translation all in an eIF2alpha-independent manner. To identify the responsible gene, proviral constructs with individually deleted auxiliary and regulatory viral genes were expressed in HeLa cells. Among the proviral deletion constructs tested, two were found to be unable to block SG assembly: pNLXX, and Rev-. Both of these constructs share one important similarity in that they all do not express the main structural protein Gag due either to the presence of premature stop codons (pNLXX) or the nuclear retention of the genomic RNA that encodes Gag (Rev-). To directly test the hypothesis that the Gag polyprotein alone blocks stress granule assembly, we transfected cells with various mammalian expression constructs of Gag. These all prevented SG assembly. We went on to further map the responsible domain of Gag to the N-terminus of the Capsid domain, specifically to two amino acids. In conclusion, HIV-1 Gag prevents SG assembly via the N-terminus of Capsid and may confer to the virus a replicative advantage during times of stress.Le génome du virus de l'immunodéficience humaine 1 (VIH-1) comprend les protéines structurales principales Gag, Gag/Pol (structurale et enzymatique), auxiliaires (Vpr, Vpu, Vif) et régulatrices (Tat, Rev, Nef). L'expression de toutes ces protéines virales augmente la réplication et le potentiel pathogène du virus. Nos résultats précédents montrent que l'assemblage des granules de stress par un processus dépendant de eIF2alpha peut être bloqué par l'expression des gènes du VIH-1. Les granules de stress sont des sites de triage d'ARN sans traduction et elles peuvent être visualisées grâce à l'accumulation d'ARN cytoplasmique et au recrutement de plusieurs protéines cytoplasmiques (TIA/R, G3BP and eIF3, par exemple). Les résultats présentés ici montrent comment le VIH-1 empêche la formation des granules de stress et caractérisent les facteurs impliqués dans ce processus. Pour provoquer la formation de granules de stress, le composé Pateamine A a été utilisé. Pateamine A cause l'activation de eIF4A, qui provoque la dissolution du complexe eIF4F et arrête la traduction par un processus indépendant de eIF2alpha. Pour identifier le gène du VIH qui empêche la formation de granules de stress, des constructions provirales individuellement déficientes dans un gène auxiliaire ou régulateur ont été exprimées dans des cellules HeLa. Parmi les constructions provirales testées, deux étaient incapables de prévenir la formation de granules de stress: pNLXX, et Rev-. Ces deux constructions ne peuvent pas exprimer la protéine structurale Gag, soit à cause de la présence de codons stop prématurés (pNL-XX), soit à cause de la rétention dans le noyau de l'ARN viral codant pour Gag (Rev-). Pour déterminer si la polyprotéine Gag est suffisante pour empêcher la formation de granules de stress, des cellules HeLa ont été transfectées avec plusieurs différents vecteurs exprimant Gag. Tous ont empêché la formation de granules de stress. De plus, nous avons déterminé que l'extrémité N-terminale du domaine de Capside de Gag est responsable de cette inhibition. En particulier, l'activité anti-granules de stresse réside dans deux acides aminés. Ainsi, la protéine Gag du VIH-1 empêche la formation de granules de stress via l'extrémité N-terminale de la Capside et pourrait donner au virus un avantage réplicatif dans des conditions de stress

HIV and the Gut Microbiota, Partners in Crime: Breaking the Vicious Cycle to Unearth New Therapeutic Targets

The gut microbiota plays a key role in health and immune system education and surveillance. The delicate balance between microbial growth and containment is controlled by the immune system. However, this balance is disrupted in cases of chronic viral infections such as HIV. This virus is capable of drastically altering the immune system and gastrointestinal environment leading to significant changes to the gut microbiota and mucosal permeability resulting in microbial translocation from the gut into the peripheral blood. The changes made locally in the gut have far-reaching consequences on the other organs of the body starting in the liver, where microbes and their products are normally filtered out, and extending to the blood and even brain. Microbial translocation and their downstream effects such as increased indolamine 2,3-dioxygenase (IDO) enzyme expression and activity create a self-sustaining feedback loop which enhances HIV disease progression and constitute a vicious cycle of inflammation and immune activation combining viral and bacterial factors. Understanding this self-perpetuating cycle could be a key element in developing new therapies aimed at the gut microbiota and its fallout after infection

Clinical Relevance of Kynurenine Pathway in HIV/AIDS: An Immune Checkpoint at the Crossroads of Metabolism and Inflammation

Tryptophan degradation along the kynurenine pathway is associated with a wide variety of pathophysiological processes, of which tumor tolerance and immune dysfunction in several chronic viral infections including HIV are well known. The kynurenine pathway is at the crossroads of metabolism and immunity and plays an important role in inflammation while also playing an opposing role in the control of acute and chronic infections. In this review we have summarized findings from recent studies reporting modulation of tryptophan degrading the kynurenine pathway in the context of HIV infection. This immuno-metabolic pathway is modulated by three distinct inducible enzymes: indoleamine 2,3-dioxygenase 1 and 2 and tryptophan 2,3-dioxygenase. Increased expression of these enzymes by antigen-presenting cells leads to local or systemic tryptophan depletion, resulting in a mechanism of defense against certain microorganisms. Conversely, it can also lead to immunosuppression by antigen-specific T-cell exhaustion and recruitment of T regulatory cells. Recently, among these enzymes, indoleamine 2,3-dioxygenase 1 has been recognized to be an immune response checkpoint that plays an important role in HIV immune dysfunction, even in the context of antiretroviral therapy. In addition to the activation of the kynurenine pathway by HIV proteins Tat and Nef, the tryptophan-degrading bacteria present in the intestinal flora have been associated with dysfunction of gut mucosal CD4 Th17/Th22 cells, leading to microbial translocation and creating a systemic kynurenine pathway activation cycle. This self-sustaining feedback loop has deleterious effects on disease progression and on neurocognitive impairment in HIV-infected patients. Therapy designed to break the vicious cycle of induced tryptophan degradation is warranted to revert immune exhaustion in HIV-infected persons

HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking

Influence of Hepatitis C Virus Sustained Virological Response on Immunosuppressive Tryptophan Catabolism in ART-Treated HIV/HCV Coinfected Patients

Background: We previously reported an association between tryptophan (Trp) catabolism and immune dysfunction in HIV monoinfection. Coinfection with HIV is associated with more rapid evolution of hepatitis C virus (HCV)-associated liver disease despite antiretroviral therapy (ART), possibly due to immune dysregulation. We hypothesized that liver fibrosis in HIV/HCV coinfection would be associated with immune dysfunction and alterations in Trp metabolism. Methods: Trp catabolism and inflammatory soluble markers were assessed in plasma samples from ART-treated HIV/HCV-coinfected patients (n = 90) compared with ART-treated HIV-monoinfected patients and noninfected subjects. Furthermore, 17 additional coinfected patients with sustained virological response (SVR) were assessed longitudinally 6 months after completion of interferon-alpha/ribavirin treatment. Results: HIV/HCV patients had higher Trp catabolism compared with HIV-monoinfected and healthy individuals. Elevated kynurenine levels in HIV/HCV patients with liver fibrosis correlated with the prognostic aspartate aminotransaminase to platelet ratio (APRI scores) and insulin levels. Furthermore, HIV/HCV patients had elevated levels of disease progression markers interleukin-6 and induced protein 10 and shared similar levels of markers of microbial translocation (intestinal fatty acid-binding protein, soluble CD14 and lipopolysaccharide-binding protein) compared with HIV-monoinfected and healthy individuals. Successful HCV treatment improved APRI score and markers of disease progression and microbial translocation although elevated Trp catabolism remained unchanged 6 months after SVR. Conclusion: ART-treated HIV/HCV-coinfected patients had elevated immunosuppressive Trp catabolism when compared with monoinfected HIV-treated patients, which did not normalize after SVR. These findings suggest that a necroinflammatory liver syndrome persists through inflammation by Trp catabolism after 6 month of SVR