Methamphetamine Enhances HIV-Induced Aberrant Proliferation of Neural Progenitor Cells via the FOXO3-Mediated Mechanism.

Maintaining an intact pool of neural progenitor cells (NPCs) is crucial for generating new and functionally active neurons. Methamphetamine (METH) can exacerbate the HIV-induced deficit of adult neurogenesis; however, potential mechanisms of this influence are still poorly understood. In the present study, we present evidence that chronic exposure to METH combined with brain infection by EcoHIV results in enhanced proliferation of NPCs in the subventricular zone (SVZ) in mice. This effect was long-lasting as it was preserved ex vivo in NPCs isolated from the exposed mice over several passages in the absence of additional treatments. Increased proliferation in response to METH plus HIV was associated with dysregulation of cyclin B1 and cyclin D. Transcriptomic studies indicated that 27 out of the top 30 differentially expressed genes in response to METH plus EcoHIV were targets of the forkhead box O transcriptional factor (FOXO) and primarily FOXO3. Additional ex vivo studies and in vitro experiments using human NPCs exposed to METH and infected with HIV revealed upregulation of the CXCL12-CXCR4 axis, leading to activation of downstream pAkt and pErk, the pathways that can phosphorylate FOXO3 and force its exports from the nuclei into the cytoplasm. Indeed, nuclear expulsion of FOXO3 was demonstrated both in mice exposed to METH and infected with EcoHIV and in cell cultures of human NPCs. These results provide novel information that exposure to METH combined with HIV infection can induce aberrant proliferation of SVZ-derived NPCs and identifies CXCL12-CXCR4-Akt-1-mediated phosphorylation of FOXO3 as the mechanism responsible for this effect.

HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals.

HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals

HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

HIV-1 genetic diversity and divergence and its correlation with disease progression among antiretroviral naïve recently infected individuals

HIV-1 genetic diversity evolution was deeply characterized during the first year of infection among recently-infected patients using deep sequencing technology and correlated with disease progression surrogate markers. RNA and DNA samples from twenty-five individuals (13 female) encoding the protease and reverse transcriptase regions of the pol gene, and the V3 region of the env gene were evaluated at recent infection and during established infection. Infection by a unique HIV-1 strain was inferred in 70.1% of the individuals, with no differences between genders. Infections by multiple strains were associated with higher viral loads and faster CD4+ T cell declines. Either low or high levels of viral loads accompanied low levels of genetic diversity and lower selective pressure. With massive sequence data from 3 distinct genomic HIV-1 regions from plasma and PBMCs over time, we propose a model for HIV-1 genetic diversity, which correlates to basal viral loads of patients.

Selective Disruption of the Blood-Brain Barrier by Zika Virus.

The blood-brain barrier (BBB) selectively regulates the cellular exchange of macromolecules between the circulation and the central nervous system (CNS). Here, we hypothesize that Zika virus (ZIKV) infects the brain via a disrupted BBB and altered expression of tight junction (TJ) proteins, which are structural components of the BBB. To assess this hypothesis, in vitro and in vivo studies were performed using three different strains of ZIKV: Honduras (ZIKV-H), Puerto Rico (ZIKV-PR), and Uganda (ZIKV-U). Primary human brain microvascular endothelial cells (BMECs) were productively infected by all studied ZIKV strains at MOI 0.01, and were analyzed by plaque assay, immunofluorescence for NS1 protein, and qRT-PCR at 2 and 6 days post-infection (dpi). Compared to mock-infected controls, expression level of ZO-1 was significantly upregulated in ZIKV-H-infected BMECs, while occludin and claudin-5 levels were significantly downregulated in BMECs infected by all three studied viral strains. Interestingly, BMEC permeability was not disturbed by ZIKV infection, even in the presence of a very high viral load (MOI 10). All studied ZIKV strains productively infected wild-type C57BL/J mice after intravenous infection with 107 PFU. Viral load was detected in the plasma, spleen, and brain from 1 to 8 dpi. Peak brain infection was observed at 2 dpi; therefore, TJ protein expression was assessed at this time point. Claudin-5 was significantly downregulated in ZIKV-U-infected animals and the BBB integrity was significantly disturbed in ZIKV-H-infected animals. Our results suggest that ZIKV penetrates the brain parenchyma early after infection with concurrent alterations of TJ protein expression and disruption of the BBB permeability in a strain-dependent manner.

Targeting the HIV-infected brain to improve ischemic stroke outcome.

HIV-associated cerebrovascular events remain highly prevalent even in the current era of antiretroviral therapy (ART). We hypothesize that low-level HIV replication and associated inflammation endure despite antiretroviral treatment and affect ischemic stroke severity and outcomes. Using the EcoHIV infection model and the middle cerebral artery occlusion as the ischemic stroke model in mice, we present in vivo analysis of the relationship between HIV and stroke outcome. EcoHIV infection increases infarct size and negatively impacts tissue and functional recovery. Ischemic stroke also results in an increase in EcoHIV presence in the affected regions, suggesting post-stroke reactivation that magnifies pro-inflammatory status. Importantly, ART with a high CNS penetration effectiveness (CPE) is more beneficial than low CPE treatment in limiting tissue injury and accelerating post-stroke recovery. These results provide potential insight for treatment of HIV-infected patients that are at risk of developing cerebrovascular disease, such as ischemic stroke.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression.

Objectives: The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Methods: Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Results: Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies >20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies <14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+ T cell count decay. Conclusions: Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Methamphetamine increases HIV infectivity in neural progenitor cells.

HIV-1 infection and methamphetamine (METH) abuse frequently occur simultaneously and may have synergistic pathological effects. Although HIV-positive/active METH users have been shown to have higher HIV viral loads and experience more severe neurological complications than non-users, the direct impact of METH on HIV infection and its link to the development of neurocognitive alternations are still poorly understood. In the present study, we hypothesized that METH impacts HIV infection of neural progenitor cells (NPCs) by a mechanism encompassing NFκB/SP1-mediated HIV LTR activation. Mouse and human NPCs were infected with EcoHIV (modified HIV virus infectious to mice) and HIV, respectively, in the presence or absence of METH (50 or 100 µm). Pretreatment with METH, but not simultaneous exposure, significantly increased HIV production in both mouse and human NPCs. To determine the mechanisms underlying these effects, cells were transfected with different variants of HIV LTR promoters and then exposed to METH. METH treatment induced transcriptional activity of the HIV LTR promotor, an effect that required both NFκB and SP1 signaling. Pretreatment with METH also decreased neuronal differentiation of HIV-infected NPCs in both in vitro and in vivo settings. Importantly, NPC-derived daughter cells appeared to be latently infected with HIV. This study indicates that METH increases HIV infectivity of NPCs, through the NFκB/SP1-dependent activation of the HIV LTR and with the subsequent alterations of NPC neurogenesis. Such events may underlie METH- exacerbated neurocognitive dysfunction in HIV-infected patients.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression

Objectives: The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Methods: Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Results: Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies.20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies,14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+T cell count decay. Conclusions: Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression

Objectives: The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Methods: Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Results: Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies.20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies,14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+T cell count decay. Conclusions: Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Pace of Coreceptor Tropism Switch in HIV-1-Infected Individuals after Recent Infection.

HIV-1 entry into target cells influences several aspects of HIV-1 pathogenesis, including viral tropism, HIV-1 transmission and disease progression, and response to entry inhibitors. The evolution from CCR5- to CXCR4-using strains in a given human host is still unpredictable. Here we analyzed timing and predictors for coreceptor evolution among recently HIV-1-infected individuals. Proviral DNA was longitudinally evaluated in 66 individuals using Geno2pheno[coreceptor] Demographics, viral load, CD4+ and CD8+ T cell counts, CCR5Δ32 polymorphisms, GB virus C (GBV-C) coinfection, and HLA profiles were also evaluated. Ultradeep sequencing was performed on initial samples from 11 selected individuals. A tropism switch from CCR5- to CXCR4-using strains was identified in 9/49 (18.4%) individuals. Only a low baseline false-positive rate (FPR) was found to be a significant tropism switch predictor. No minor CXCR4-using variants were identified in initial samples of 4 of 5 R5/non-R5 switchers. Logistic regression analysis showed that patients with an FPR of >40.6% at baseline presented a stable FPR over time whereas lower FPRs tend to progressively decay, leading to emergence of CXCR4-using strains, with a mean evolution time of 27.29 months (range, 8.90 to 64.62). An FPR threshold above 40.6% determined by logistic regression analysis may make it unnecessary to further determine tropism for prediction of disease progression related to emergence of X4 strains or use of CCR5 antagonists. The detection of variants with intermediate FPRs and progressive FPR decay over time not only strengthens the power of Geno2pheno in predicting HIV tropism but also indirectly confirms a continuous evolution from earlier R5 variants toward CXCR4-using strains.IMPORTANCE The introduction of CCR5 antagonists in the antiretroviral arsenal has sparked interest in coreceptors utilized by HIV-1. Despite concentrated efforts, viral and human host features predicting tropism switch are still poorly understood. Limited longitudinal data are available to assess the influence that these factors have on predicting tropism switch and disease progression. The present study describes longitudinal tropism evolution in a group of recently HIV-infected individuals to determine the prevalence and potential correlates of tropism switch. We demonstrated here that a low baseline FPR determined by the Geno2pheno[coreceptor] algorithm can predict tropism evolution from CCR5 to CXCR4 coreceptor use.

Emergence of CD4 independence envelopes and astrocyte infection in R5 simian-human immunodeficiency virus model of encephalitis.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) is characterized by replication in macrophages or brain microglia that express low levels of the CD4 receptor and is the cause of HIV-associated dementia and related cognitive and motor disorders that affect 20 to 30% of treatment-naive patients with AIDS. Independent viral envelope evolution in the brain has been reported, with the need for robust replication in resident CD4(low) cells, as well as CD4-negative cells, such as astrocytes, proposed as a major selective pressure. We previously reported giant-cell encephalitis in subtype B and C R5 simian-human immunodeficiency virus (SHIV)-infected macaques (SHIV-induced encephalitis [SHIVE]) that experienced very high chronic viral loads and progressed rapidly to AIDS, with varying degrees of macrophage or microglia infection and activation of these immune cells, as well as astrocytes, in the CNS. In this study, we characterized envelopes (Env) amplified from the brains of subtype B and C R5 SHIVE macaques. We obtained data in support of an association between severe neuropathological changes, robust macrophage and microglia infection, and evolution to CD4 independence. Moreover, the degree of Env CD4 independence appeared to correlate with the extent of astrocyte infection in vivo. These findings further our knowledge of the CNS viral population phenotypes that are associated with the severity of HIV/SHIV-induced neurological injury and improve our understanding of the mechanism of HIV-1 cellular tropism and persistence in the brain. IMPORTANCE: Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes in the brain has been suggested to be important in HIV persistence and neuropathogenesis but has not been definitively demonstrated in an animal model of HIV-induced encephalitis (HIVE). Here, we describe a new nonhuman primate (NHP) model of R5 simian-human immunodeficiency virus (SHIV)-induced encephalitis (SHIVE) with several classical HIVE features that include astrocyte infection. We further show an association between severe neuropathological changes, robust resident microglia infection, and evolution to CD4 independence of viruses in the central nervous system (CNS), with expansion to infection of truly CD4-negative cells in vivo. These findings support the use of the R5 SHIVE models to study the contribution of the HIV envelope and viral clades to neurovirulence and residual virus replication in the CNS, providing information that should guide efforts to eradicate HIV from the body.

The number and genetic relatedness of transmitted/founder virus impact clinical outcome in vaginal R5 SHIVSF162P3N infection.

BACKGROUND: Severe genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys. RESULTS: We found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1-2 T/F viruses found in the CPs and 2-4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter. CONCLUSION: Our study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.