Polymorphism in HIV-1 dependency factor PDE8A affects gene expression and HIV-1 replication in primary macrophages

The limited size of the human immunodeficiency virus 1 (HIV-1) genome and the small number of proteins it encodes make the virus highly dependent on host proteins for its replication. Four genome-wide RNAi screens have recently identified a large number of HIV-1 dependency factors (HDFs), with the majority of these proteins never before associated with HIV-1 replication. Recently, we reported more than 3 log variation in the ability of HIV-1 to replicate in monocyte derived macrophages (MDM) derived from \u3e4OO healthy seronegative blood donors. In our present study we determined whether single nucleotide polymorphisms (SNPs) in the genes encoding the newly identified HDFs were associated with this variation in HIV-1 replication

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells.

Unlike resting CD4+ T cells, activated CD4+T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4+T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4+T cells

Identification of a new genotype of Torque Teno Mini virus

Although human torque teno viruses (TTVs) were first discovered in 1997, still many associated aspects are not clarified yet. The viruses reveal a remarkable heterogeneity and it is possible that some genotypes are more pathogenic than others. The identification of all genotypes is essential to confirm previous pathogenicity data, and an unbiased search for novel viruses is needed to identify TTVs that might be related to disease. The virus discovery technique VIDISCA-454 was used to screen serum of 55 HIV-1 positive injecting drug users, from the Amsterdam Cohort Studies, in search for novel blood-blood transmittable viruses which are undetectable via normal diagnostics or panvirus-primer PCRs. A novel torque teno mini virus (TTMV) was identified in two patients and the sequence of the full genomes were determined. The virus is significantly different from the known TTMVs ( <40% amino acid identity in ORF1), yet it contains conserved characteristics that are also present in other TTMVs. The virus is chronically present in both patients, and these patients both suffered from a pneumococcal pneumonia during follow up and had extremely low B-cells counts. We describe a novel TTMV which we tentatively named TTMV-13. Further research is needed to address the epidemiology and pathogenicity of this novel viru

Virological and immunological correlates of HIV posttreatment control after temporal antiretroviral therapy during acute HIV infection

Objective:People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary posttreatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC.Design/methods:Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay, and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T cells and HIV-specific T-cell responses were analyzed by flow cytometry.Results:The case presented with AHI and a plasma viral load of 2.7 million copies/ml. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption, HIV-DNA in CD4+T cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8+T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44 : 02 restricted gag-epitope, which was associated with decreased replication.Conclusion:We describe an exceptional case of PTC, which is likely associated with sustained potent gag-specific CD8+T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control

Polymorphisms in the Regulatory Region of the Cyclophilin A Gene Influence the Susceptibility for HIV-1 Infection

Background: Previous studies have demonstrated an association between polymorphisms in the regulatory regions of Cyclophilin A (CypA) and susceptibility to both HIV-1 infection and disease progression. Here we studied whether these polymorphisms are associated with susceptibility to HIV-1 infection and disease progression in the Amsterdam Cohort on HIV-1 infection and AIDS (ACS) in a group of men having sex with men (MSM) and drug users (DU). Methodology/Principal Findings: We screened participants of the ACS for the C1604G and A1650G polymorphisms in the regulatory regions of CypA. The prevalence of the 1650G allele was significantly higher in high risk seronegative MSM than in HIV-1 infected MSM. However, C1604G or A1650G were not associated with the clinical course of infection in MSM of the ACS. Interestingly, participants of the ACS-DU who carried the 1604G allele showed a significantly accelerated progression when viral RNA load above 10 4.5 copies per ml plasma was used as an endpoint in survival analysis. Conclusion/Significance: The results obtained in this study suggest that the A1650G polymorphism in the regulatory region of the CypA gene may be associated with protection from HIV-1 infection, while the 1604G allele may have a wea

Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages

Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophage

Identification of a Novel HBV Encoded miRNA Using Next Generation Sequencing

Hepatitis B Virus (HBV) encoded miRNAs were previously described and suggested to play a role in HBV replication and pathogenesis. In this study we aim to identify novel HBV encoded miRNAs in plasma and liver tissue samples from chronic hepatitis B (CHB) patients and determine their role in CHB pathogenesis and HBV replication. RNA next generation sequencing was performed on plasma and liver tissue samples from ten CHB patients and uninfected controls. The interaction of the potential miRNA-like structures with the RNA-induced silencing complex (RISC) was determined using RNA immunoprecipitation. Expression levels of the HBV encoded miRNAs were measured in liver tissue samples derived from a conformation cohort. The effect of HBV encoded miRNAs overexpression on HBV replication, expression of predicted target genes, and induction of interferon stimulated genes in cell lines were assessed. Three potential miRNA-like structures transcribed by HBV were identified in liver tissue, of which one miRNA, HBV-miR-6, was recognized using RISC. HBV-miR-6 expression was demonstrated in liver tissue samples from 52 of the 87 CHB patients. HBV-miR-6 levels correlated with hepatic HBV-DNA and plasma HBsAg levels. Overexpression of HBV-miR-6 in vitro did not affect HBV replication, and predicted both target genes expression and interferon stimulated genes expression after stimulation. A potential novel HBV encoded miRNA was identified and validated in liver tissue from CHB patients. It is suggested that HBV-miR-6 may play a role in the process of viral excretion or particle formation in vivo

Altered EBV viral load setpoint after HIV seroconversion is in accordance with lack of predictive value of EBV load for the occurrence of AIDS-related non-Hodgkin lymphoma

In contrast to the situation in the post-transplant setting, in HIV-infected individuals an elevated EBV load is not predictive of EBV-related malignancies. To study whether a high EBV load is already a normal situation early in HIV infection and is not related to a decrease in immune function over time, we investigated EBV load and EBV-specific CD8(+) T cells similar to1 year before and I year after HIV seroconversion. EBV load significantly increased after HIV seroconversion from 205 to 1002 copies/10(6) PBMC (p <0.001), whereas no further increase in EBV load was observed between 1 and 5 years after HIV seroconversion (median, 1827-2478 copies/10(6) PBMC; p = 0.530). Interestingly, the absolute number of EBV lytic epitope, RAKFKQLL-specific CD8(+) T cells increased over HIV seroconversion (4.78 to 9.54/mul; p = 0.011). Furthermore, the fraction of CD27-negative effector, RAK-specific CD8(+) T cells tended to increase (from 12.2 to 17.31% CD27(-); p = 0.051), in accordance with Ag-driven differentiation. In conclusion, both virological and immunological data support the idea that a new EBV viral setpoint is reached early in HIV infection, probably by EBV reactivation, as suggested by the preferential increase in EBV lytic epitope-specific CD8(+) T cells. These data may thus help to explain the lack of predictive value of EBV load for the occurrence of AIDS-related lymphom

Novel method for detection of virus-specific CD41+ T cells indicates a decreased EBV-specific CD4+ T cell response in untreated HIV-infected subjects

A lower function of EBV-specific CD8(+) T cells in HIV-infected subjects could be related to a lack of specific CD4(+) T cell help. Therefore, we studied EBV-specific CD4(+) T cells in both healthy donors and untreated or highly active antiretroviral therapy (HAART)-treated HIV-seropositive homosexual men. To this end, PBMC were stimulated with overlapping peptide pools from a latent and a lytic EBV protein, EBV nuclear antigen (EBNA)1 and EBV lytic-switch protein ZEBRA (BZLF1), respectively. EBV-specific CD4(+) T cell frequencies measured directly ex vivo were low. To measure EBV-specific memory CD4(+) T cells, capable of both expansion and IFN-gamma production upon antigenic challenge, we developed a specific and reproducible assay, combining ex vivo expansion of specific T cells with flow cytometric analysis of IFN-gamma production. Untreated HIV-infected individuals had a lower CD4(+) T cell response to both EBNA1 and BZLF1 as compared to healthy EBV carriers and HAART-treated HIV-positive subjects. This suggests loss of EBV-specific CD4(+) T cells due to HIV infection, while HAART might restore this response. In addition, we found an increase in the EBNA1-specific CD8(+) T cell response in HAART-treated subjects. Interestingly, numbers of EBV-specific CD4(+) and CD8(+) T cells were inversely correlated with EBV viral load, suggesting an important role also for EBV-specific CD4(+) T cells in the control of EBV infectio