HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression

<p>Abstract</p> <p>Background</p> <p>RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression.</p> <p>Results</p> <p>Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.</p> <p>Conclusion</p> <p>The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.</p

Live Attenuated Rev-Independent Nef¯SIV Enhances Acquisition of Heterologous SIVsmE660 in Acutely Vaccinated Rhesus Macaques

Background: Rhesus macaques (RMs) inoculated with live-attenuated Rev-Independent Nef¯ simian immunodeficiency virus (Rev-Ind Nef¯SIV) as adults or neonates controlled viremia to undetectable levels and showed no signs of immunodeficiency over 6-8 years of follow-up. We tested the capacity of this live-attenuated virus to protect RMs against pathogenic, heterologous SIVsmE660 challenges. Methodology/Principal Findings Three groups of four RM were inoculated with Rev-Ind Nef¯SIV and compared. Group 1 was inoculated 8 years prior and again 15 months before low dose intrarectal challenges with SIVsmE660. Group 2 animals were inoculated with Rev-Ind Nef¯SIV at 15 months and Group 3 at 2 weeks prior to the SIVsmE660 challenges, respectively. Group 4 served as unvaccinated controls. All RMs underwent repeated weekly low-dose intrarectal challenges with SIVsmE660. Surprisingly, all RMs with acute live-attenuated virus infection (Group 3) became superinfected with the challenge virus, in contrast to the two other vaccine groups (Groups 1 and 2) (P=0.006 for each) and controls (Group 4) (P=0.022). Gene expression analysis showed significant upregulation of innate immune response-related chemokines and their receptors, most notably CCR5 in Group 3 animals during acute infection with Rev-Ind Nef¯SIV. Conclusions/Significance: We conclude that although Rev-Ind Nef¯SIV remained apathogenic, acute replication of the vaccine strain was not protective but associated with increased acquisition of heterologous mucosal SIVsmE660 challenges

Coinfection with Schistosoma mansoni Reactivates Viremia in Rhesus Macaques with Chronic Simian-Human Immunodeficiency Virus Clade C Infection

We tested the hypothesis that helminth parasite coinfection would intensify viremia and accelerate disease progression in monkeys chronically infected with an R5 simian-human immunodeficiency virus (SHIV) encoding a human immunodeficiency virus type 1 (HIV-1) clade C envelope. Fifteen rhesus monkeys with stable SHIV-1157ip infection were enrolled into a prospective, randomized trial. These seropositive animals had undetectable viral RNA and no signs of immunodeficiency. Seven animals served as virus-only controls; eight animals were exposed to Schistosoma mansoni cercariae. From week 5 after parasite exposure onward, coinfected animals shed eggs in their feces, developed eosinophilia, and had significantly higher mRNA expression of the T-helper type 2 cytokine interleukin-4 (P = 0.001) than animals without schistosomiasis. Compared to virus-only controls, viral replication was significantly increased in coinfected monkeys (P = 0.012), and the percentage of their CD4(+) CD29(+) memory cells decreased over time (P = 0.05). Thus, S. mansoni coinfection significantly increased viral replication and induced T-cell subset alterations in monkeys with chronic SHIV clade C infection

Vaccination strategy with Rev-Ind Nef¯SIV and SIVsmE660 challenges.

<p>RMs in Groups 1(low-dose challenge group) 5 and (high-dose only challenge group) had been vaccinated with Rev-Ind Nef¯SIV >8 yrs ago and boosted along with Group 2 monkeys; RMs in Group 2 were vaccinated with Rev-Ind Nef¯SIV 15 months prior to SIVsmE660 low-dose challenges, RMs in Group 3 were vaccinated with Rev-Ind Nef¯SIV 2 weeks prior to SIVsmE660 low-dose challenges; Group 4 consisted of naïve RMs that served as controls for SIVsmE660 challenges; Groups 6, served as control unvaccinated group was challenged with a high-dose of SIVsmE660 only and Group 7 received only Rev-Ind Nef¯SIV and was followed for 2 weeks. Groups 1-4 received the maximum of 17 weekly low-dose i.r. challenges with SIVsmE660 (300 TCID₅₀ as measured by TZM-bl assay). RMs that did not become systemically infected after the 17 low-dose challenges (boxed names) were rechallenged i.r. with a single high dose of SIVsmE660 (990 TCID₅₀) and became infected (rechallenged Group 1-2 and high-dose control Group 6). Blue indicates the live attenuated vaccine strain (Rev-Ind Nef¯SIV). A fifth monkey (RMw-6) did not fulfill the criteria of “vaccine take” (transient low-dose Rev-Ind Nef¯SIV infection with vRNA of 1080 copies/ml at 2 weeks post-vaccination, the expected time of peak vaccine virus viremia); therefore, data from RWm-6 were not included in the analysis. Thin red arrows stand for the weekly low-dose i.r. exposures to the challenge virus, heterologous SIVsmE660; the thick red arrows represent single high-dose i.r. challenges with SIVsmE660.</p

Network pathway analysis.

<p>Network pathway analyses were performed using the Ingenuity Pathway Analysis Network tool. Node colors show the average gene expression levels of RMs vaccinated with Rev-Ind Nef¯SIV relative to the average before vaccination (red, relative increase in expression; green, relative decrease in expression). CCR5, CCL3 and other signaling molecules were upregulated and were directly and indirectly involved with other immune system pathways. The numbers underneath the nodes are 1) log₂-fold change, and 2) FDR-adjusted <i>P</i> values.</p

Plasma vRNA loads after low-dose (A-B; D-E; G-H; J-K) or high-dose (M-N; P-Q) SIVsmE660 challenges.

<p>Horizontal dashed lines in all panels represent the limit of detection of the RT-PCR assay (50 vRNA copies/ml) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075556#B32" target="_blank">32</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075556#B33" target="_blank">33</a>]. RMs in boxes were entered into the single high-dose challenge with SIVsmE660 (thick arrow) shown in M-N and P-Q. The panels in (C, F, I, L, O, R) are mean vRNA loads of each group. Blue, indicates vRNA loads for the live attenuated vaccine strain (Rev-Ind Nef¯SIV), purple indicates vRNA loads both vaccine + challenge virus, and the red line indicates challenge virus-specific vRNA loads.</p

vRNA load measurements, principal component analysis (PCA) and heatmap analysis.

<p>(A) vRNA loads were measured at 2 weeks after Rev-Ind Nef¯SIV vaccination (Group 7; 3 RMs) The RMs were subsequently euthanized to collect rectal tissues and PBMC. (B) PCA was performed on log₂ expression measures for autologous PBMC and rectal biopsy specimens collected before and two weeks after inoculation with the live attenuated vaccine strain, Rev-Ind Nef¯SIV. Infected and naïve PBMC samples were centered and grouped together, indicating that the samples were nearly homogenous. In contrast, we observed heterogeneity in rectal tissues. (C) Hierarchical clustering of differentially expressed genes in PBMC. Supervised analysis identified approximately 100 differentially expressed genes between PBMC of the RMs collected before and after vaccination using a significance threshold of a false discovery rate) <5% and log₂ fold-change >1 was analyzed using average-linkage hierarchical clustering with a Pearson correlation coefficient distance metric. We noted a clear upregulation of expression of a set of chemokines and chemokine receptors, most notably CCL3, and CCL5 (highlighted).</p