Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.     

Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis

HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ～10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis- and trans-acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

Comparative analysis of Rev function in human immunodeficiency virus types 1 and 2.

The Rev proteins of the related but distinct human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation for this observation is that HIV-2 Rev is unable to interact with the HIV-1 Rev-response element (RRE1). However, an analysis of the biological activity of chimeric proteins derived from HIV-1 and HIV-2 Rev reveals that this target specificity does not map to the Rev RNA binding domain but is instead primarily determined by sequences known to mediate Rev multimerization. Both HIV-1 and HIV-2 Rev are shown to bind the RRE1 in vitro with identical RNA sequence specificity. The observation that HIV-2 Rev can inhibit RRE1-dependent HIV-1 Rev function in trans indicates that the direct interaction of HIV-2 Rev with the RRE1 also occurs in vivo. These data suggest that HIV-2 Rev forms a protein-RNA complex with the RRE1 that leads to only minimal Rev activity. It is hypothesized that this low level of Rev function results from the incomplete and/or aberrant multimerization of HIV-2 Rev on this heterologous RNA target sequence.     

Estimating the Impact of Plasma HIV-1 RNA Reductions on Heterosexual HIV-1 Transmission Risk

Background

The risk of sexual transmission of HIV-1 is strongly associated with the level of HIV-1 RNA in plasma making reduction in HIV-1 plasma levels an important target for HIV-1 prevention interventions. A quantitative understanding of the relationship of plasma HIV-1 RNA and HIV-1 transmission risk could help predict the impact of candidate HIV-1 prevention interventions that operate by reducing plasma HIV-1 levels, such as antiretroviral therapy (ART), therapeutic vaccines, and other non-ART interventions.

Methodology/Principal Findings

We use prospective data collected from 2004 to 2008 in East and Southern African HIV-1 serodiscordant couples to model the relationship of plasma HIV-1 RNA levels and heterosexual transmission risk with confirmation of HIV-1 transmission events by HIV-1 sequencing. The model is based on follow-up of 3381 HIV-1 serodiscordant couples over 5017 person-years encompassing 108 genetically-linked HIV-1 transmission events. HIV-1 transmission risk was 2.27 per 100 person-years with a log-linear relationship to log₁₀ plasma HIV-1 RNA. The model predicts that a decrease in average plasma HIV-1 RNA of 0.74 log₁₀ copies/mL (95% CI 0.60 to 0.97) reduces heterosexual transmission risk by 50%, regardless of the average starting plasma HIV-1 level in the population and independent of other HIV-1-related population characteristics. In a simulated population with a similar plasma HIV-1 RNA distribution the model estimates that 90% of overall HIV-1 infections averted by a 0.74 copies/mL reduction in plasma HIV-1 RNA could be achieved by targeting this reduction to the 58% of the cohort with plasma HIV-1 levels ≥4 log₁₀ copies/mL.

Conclusions/Significance

This log-linear model of plasma HIV-1 levels and risk of sexual HIV-1 transmission may help estimate the impact on HIV-1 transmission and infections averted from candidate interventions that reduce plasma HIV-1 RNA levels.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

Nonreciprocal Packaging of Human Immunodeficiency Virus Type 1 and Type 2 RNA: a Possible Role for the p2 Domain of Gag in RNA Encapsidation

The ability of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) to cross-package each other’s RNA was investigated by cotransfecting helper virus constructs with vectors derived from both viruses from which the gag and pol sequences had been removed. HIV-1 was able to package both HIV-1 and HIV-2 vector RNA. The unspliced HIV-1 vector RNA was packaged preferentially over spliced RNA; however, unspliced and spliced HIV-2 vector RNA were packaged in proportion to their cytoplasmic concentrations. The HIV-2 helper virus was unable to package the HIV-1 vector RNA, indicating a nonreciprocal RNA packaging relationship between these two lentiviruses. Chimeric proviruses based on HIV-2 were constructed to identify the regions of the HIV-1 Gag protein conferring RNA-packaging specificity for the HIV-1 packaging signal. Two chimeric viruses were constructed in which domains within the HIV-2 gag gene were replaced by the corresponding domains in HIV-1, and the ability of the chimeric proviruses to encapsidate an HIV-1-based vector was studied. Wild-type HIV-2 was unable to package the HIV-1-based vector; however, replacement of the HIV-2 nucleocapsid by that of HIV-1 generated a virus with normal protein processing which could package the HIV-1-based vector. The chimeric viruses retained the ability to package HIV-2 genomic RNA, providing further evidence for a lack of reciprocity in RNA-packaging ability between the HIV-1 and HIV-2 nucleocapsid proteins. Inclusion of the p2 domain of HIV-1 Gag in the chimera significantly enhanced packaging.     

HIV-1 rev promotes the nuclear export of unspliced and singly spliced RNAs in a mammalian cell-free export system

Rev has been shown to promote the export of HIV-1 RNAs fromXenopus oocyte nuclei, but a system to examine the direct effect of Rev on HIV-1 RNA export in mammalian somatic cells does not exist. In this report, the development of a cell-free RNA export system using COS cells is described. This system is capable of examining the movement of RNA from nuclei of COS cells transfected with an HIV-1 proviral construct into reconstituted cytosol from nontransfected cells. A reproducible preparation of nuclei free of residual cytoplasmic RNA is demonstrated. Export of RNA from these nuclei into reconstituted cell-free extracts was saturable and dependent on temperature and energy. Further validation of the system was obtained by confirming that the nuclear export of HIV-1-unspliced and partially spliced RNAs was dependent upon the expression of HIV-1 Rev and that the presence of Rev appeared to decrease the export of an HIV-1-spliced RNA. The system was also able to demonstrate that Rev did not appear to significantly enhance the export of an HIV-1 protease-containing RNA that has been shown to be dependent upon Rev for maximal expression. Consequently, the system appears useful for the examination of parameters of nuclear export of HIV-1 and cellular RNAs.     

Acceleration of Age-Associated Methylation Patterns in HIV-1-Infected Adults

Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x10^-200 and 0.47, p<1x10^-200. Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1⁺ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: β_age= 0.007088, p=2.08 x 10^-9; β_HIV= 0.099574, p=0.0011; Data set 2: β_age= 0.008762, p=1.27x 10^-5; β_HIV= 0.128649, p= 0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10^-6, odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular and molecular mechanisms. These results are an important first step for finding potential therapeutic targets and novel clinical approaches to mitigate the detrimental effects of both HIV-1-infection and aging.