|Retrovirology - Latest Articles|
Background: All lentiviruses except equine infectious anemia virus (EIVA) antagonize antiviral family APOBEC3 (A3) proteins of the host through viral Vif proteins. The mechanism by which Vif of human, simian or feline immunodeficiency viruses (HIV/SIV/FIV) suppresses the corresponding host A3s has been studied extensively. Results: Here, we determined that bovine immunodeficiency virus (BIV) and maedi-visna virus (MVV) Vif proteins utilize the Cullin (Cul)-ElonginB (EloB)-ElonginC (EloC) complex (BIV Vif recruits Cul2, while MVV Vif recruits Cul5) to degrade Bos taurus (bt)A3Z2-Z3 and Ovis aries (oa)A3Z2-Z3, respectively, via a proteasome-dependent but a CBF-?-independent pathway. Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC and oaEloC and dominant-negative mutants of btCul2 and oaCul5 could disrupt the activity of BIV and MVV Vif, respectively. While the membrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation of btA3Z2-Z3, it had minimal effects on oaA3Z2-Z3 degradation induced by MVV Vif, indicating that Zn is important for the activity of BIV Vif but not MVV Vif. Furthermore, we identified a previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC box which is critical for its degradation activity. Conclusions: A novel zinc binding loop was identified in the BIV Vif protein that is important for the E3 ubiquination activity, suggesting that the degradation of btA3Z2-Z3 by BIV and that of oaA3Z2-Z3 by MVV Vif may need host factors other than CBF-?.
Background: All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to accomplish this. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. Results: The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag?only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis. Conclusion: These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.
Background: A major immune evasion mechanism of HIV-1 is the accumulation of non-synonymous mutations in and around T cell epitopes, resulting in loss of T cell recognition and virus escape. Results: Here we analyze primary CD8+ T cell responses and virus escape in a HLA B*81 expressing subject who was infected with two T/F viruses from a single donor. In addition to classic escape through non-synonymous mutation/s, we also observed rapid selection of multiple recombinant viruses that conferred escape from T cells specific for two epitopes in Nef. Conclusions: Our study shows that recombination between multiple T/F viruses provide greater options for acute escape from CD8+ T cell responses than seen in cases of single T/F virus infection. This process may contribute to the rapid disease progression in patients infected by multiple T/F viruses.
Background: Aged individuals respond poorly to vaccination and have a higher risk of contracting infections in comparison to younger individuals; whether age impacts on the composition and function of B cell subpopulations relevant for immune responses is still controversial. It is also not known whether increased age during HIV-1 infection further synergizes with the virus to alter B cell subpopulations. In view of the increased number of HIV-1 infected patients living to high age as a result of anti-retroviral treatment this is an important issue to clarify. Results: In this work we evaluated the distribution of B cell subpopulations in young and aged healthy individuals and HIV-1 infected patients, treated and na?ve to treatment. B cell populations were characterized for the expression of inhibitory molecules (PD-1 and FcRL4) and activation markers (CD25 and CD69); the capacity of B cells to respond to activation signals through up-regulation of IL-6 expression was also evaluated. Increased frequencies of activated and tissue-like memory B cells occurring during HIV-1 infection are corrected by prolonged ART therapy. Our findings also reveal that, in spite of prolonged treatment, resting memory B cells in both young and aged HIV-1 infected patients are reduced in number, and all memory B cell subsets show a low level of expression of the activation markers CD25. Conclusions: The results of our study show that resting memory B cells in ART-treated young and aged HIV-1 infected patients are reduced in number and memory B cell subsets exhibit low expression of the activation marker CD25. Aging per se in the HIV-1 infected population does not worsen impairments initiated by HIV-1 in the memory B cell populations of young individuals.
Background: The reproducible nature of HIV-1 escape from HLA-restricted CD8+ T-cell responses allows the identification of HLA-associated viral polymorphisms ?at the population level? ? that is, via analysis of cross-sectional, linked HLA/HIV-1 genotypes by statistical association. However, elucidating their timing of selection traditionally requires detailed longitudinal studies, which are challenging to undertake on a large scale. We investigate whether the extent and relative timecourse of immune-driven HIV adaptation can be inferred via comparative cross-sectional analysis of independent early and chronic infection cohorts. Results: Similarly-powered datasets of linked HLA/HIV-1 genotypes from individuals with early (median?llt;?3 months) and chronic untreated HIV-1 subtype B infection, matched for size (N?ggt;?200/dataset), HLA class I and HIV-1 Gag/Pol/Nef diversity, were established. These datasets were first used to define a list of 162 known HLA-associated polymorphisms detectable at the population level in cohorts of the present size and host/viral genetic composition. Of these 162 known HLA-associated polymorphisms, 15% (occurring at 14 Gag, Pol and Nef codons) were already detectable via statistical association in the early infection dataset at p???0.01 (q?llt;?0.2) ? identifying them as the most consistently rapidly escaping sites in HIV-1. Among these were known rapidly-escaping sites (e.g. B*57-Gag-T242N) and others not previously appreciated to be reproducibly rapidly selected (e.g. A*31:01-associated adaptations at Gag codons 397, 401 and 403). Escape prevalence in early infection correlated strongly with first-year escape rates (Pearson?s R?=?0.68, p?=?0.0001), supporting cross-sectional parameters as reliable indicators of longitudinally-derived measures. Comparative analysis of early and chronic datasets revealed that, on average, the prevalence of HLA-associated polymorphisms more than doubles between these two infection stages in persons harboring the relevant HLA (p?llt;?0.0001, consistent with frequent and reproducible escape), but remains relatively stable in persons lacking the HLA (p?=?0.15, consistent with slow reversion). Published HLA-specific Hazard Ratios for progression to AIDS correlated positively with average escape prevalence in early infection (Pearson?s R?=?0.53, p?=?0.028), consistent with high early within-host HIV-1 adaptation (via rapid escape and/or frequent polymorphism transmission) as a correlate of progression. Conclusion: Cross-sectional host/viral genotype datasets represent an underutilized resource to identify reproducible early pathways of HIV-1 adaptation and identify correlates of protective immunity.
Background: The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat.Both vif and vpr mRNAs harbor their translational start codons within the intron bordering the non-coding leader exons 2 and 3, respectively. Intron retention relies on functional cross-exon interactions between splice sites A1 and D2 (for vif mRNA) and A2 and D3 (for vpr mRNA). More precisely, prior to the catalytic step of splicing, which would lead to inclusion of the non-coding leader exons, binding of U1 snRNP to the 5aapos; splice site (5aapos;ss) facilitates recognition of the 3aapos;ss by U2 snRNP and also supports formation of vif and vpr mRNA. Results: We identified a G run localized deep in the vpr AUG containing intron 3 (GI3-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of GI3-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of GI3-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by GI3-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of GI3-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in GI3-2, we identified an adjacent G-rich sequence (GI3-1), which was able to substitute for the inactivated GI3-2. Conclusions: The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.
Background: Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections.FindingsIn this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/?cnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24?h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected. Conclusions: Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.
Background: SAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. Recently, viral protein X (Vpx) of HIV-2/SIVsm was shown to target SAMHD1 for proteosomal degradation and led to elevation of dNTP levels, which in turn accelerated proviral DNA synthesis of lentiviruses in macrophages. Results: We investigated both time-dependent and quantitative interplays between SAMHD1 level and dNTP concentrations during multiple exposures of Vpx in macrophages. The following were observed. First, SAMHD1 level was rapidly reduced by Vpx + VLP to undetectable levels by Western blot analysis. Recovery of SAMHD1 was very slow with less than 3% of the normal macrophage level detected at day 6 post Vpx treatment and only ~30% recovered at day 14. Second, dGTP, dCTP and dTTP levels peaked at day 1 post Vpx treatment, whereas dATP peaked at day 2. However, all dNTPs rapidly decreased starting at day 3, while SAMHD1 level was below the level of detection. Third, when Vpx pretreated macrophages were re-exposed to a second Vpx treatment at day 7, we observed dNTP elevation that had faster kinetics than the first Vpx + VLP treatment. Moreover, we performed a short kinetic analysis of the second Vpx treatment to find that dATP and dGTP levels peaked at 8 hours post secondary VLP treatment. dGTP peak was consistently higher than the primary, whereas peak dATP concentration was basically equivalent to the first Vpx + VLP treatment. Lastly, HIV-1 replication kinetics were faster in macrophages treated after the secondary Vpx treatments when compared to the initial single Vpx treatment. Conclusion: This study reveals that a very low level of SAMHD1 sufficiently modulates the normally low dNTP levels in macrophages and proposes potential diverse mechanisms of Vpx-mediated dNTP regulation in macrophages.
The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.
Background: The widespread use of highly effective, combination antiretroviral therapy (cART) has led to a significant reduction in the incidence of HIV-associated dementia (HAD). Despite these advances, the prevalence of HIV-1 associated neurocognitive disorders (HANDs) has been estimated at approximately 40%-50%. In the cART era, the majority of this disease burden is represented by asymptomatic neurocognitive impairment and mild neurocognitive disorder (ANI and MND respectively). Although less severe than HAD, these diagnoses carry with them substantial morbidity. Results: In this cross-sectional study, single genome amplification (SGA) was used to sequence 717 full-length HIV-1 envelope (env) clade B variants from the paired cerebrospinal fluid (CSF) and blood plasma samples of fifteen chronically infected HIV-positive individuals with normal neurocognitive performance (NCN), ANI and MND. Various degrees of compartmentalization were found across disease states and history of cART utilization. In individuals with compartmentalized virus, mean HIV-1 env population diversity was lower in the CSF than plasma-derived variants. Overall, mean V1V2 loop length was shorter in CSF-derived quasispecies when compared to contemporaneous plasma populations, and this was found to correlate with a lower mean number of N-linked glycosylation sites in this region. A number of discrete amino acid positions that correlate strongly with compartmentalization in the CSF were identified in both variable and constant regions of gp120 as well as in gp41. Correlated mutation analyses further identified that a subset of amino acid residues in these compartmentalization“hot spot” positions were strongly correlated with one another, suggesting they may play an important, definable role in the adaptation of viral variants to the CSF. Analysis of these hot spots in the context of a well-supported crystal structure of HIV-1 gp120 suggests mechanisms through which amino acid differences at the identified residues might contribute to viral compartmentalization in the CSF. Conclusions: The detailed analyses of SGA-derived full length HIV-1 env from subjects with both normal neurocognitive performance and the most common HAND diagnoses in the cART era allow us to identify novel and confirm previously described HIV-1 env genetic determinants of neuroadaptation and relate potential motifs to HIV-1 env structure and function.