Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ~1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.Weizmann Institute of Science (Postdoctoral Award for Advancing Women in Science)European Molecular Biology Organization (Long-term Fellowship)Machiah FoundationNational Science Foundation (U.S.) (Grant 0821391)National Institutes of Health (U.S.

Widespread Regulation of Translation by Elongation Pausing in Heat Shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70’s interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.National Institute of General Medical Sciences (U.S.) (NIGMS fellowship number F32GM095060)Machiah FoundationEuropean Molecular Biology Organization (EMBO long-term fellowship)Weitzmann Institute of Science (National Postdoctoral Award Program for Advancing Women in Science

Repeat Human Immunodeficiency Virus Testing by Transmission Risk Group and Rurality of Residence in North Carolina

Background Understanding of repeat human immunodeficiency virus (HIV) testing (RHT) is limited and the impact of rural residence as a potential barrier to RHT is unknown. Rural populations are of particular interest in the Southeastern United States because of their disproportionate HIV burden. Methods We used HIV surveillance data from publicly funded HIV testing sites in North Carolina to assess repeat testing by transmission risk group and residential rurality in a retrospective cohort study. Linear binomial regression models were used to estimate adjusted, 1-year cumulative incidences and cumulative incidence differences comparing RHT within transmission risk populations by level of rurality. Results In our total study population of 600,613 persons, 19,275 (3.2%) and 9567 (1.6%) self-identified as men who have sex with men (MSM) and persons who inject drugs (PWID), respectively. A small minority, 13,723 (2.3%) resided in rural ZIP codes. Men who have sex with men were most likely to repeat test (unadjusted, 1-year cumulative incidence after an initial negative test, 16.4%) compared with PWID (13.2%) and persons who did not identify as either MSM or PWID (13.6%). The greatest effect of rurality was within PWID; the adjusted, 1-year cumulative incidence of RHT was 6.4 (95% confidence interval, 1.4-11.4) percentage points higher among metropolitan versus rural PWID. Conclusions One-year cumulative incidence of RHT was low among all clients of publicly funded HIV testing sites in North Carolina, including MSM and PWID for whom annual testing is recommended. Our findings suggest a need for public health efforts to increase access to and support for RHT, particularly among rural PWID

Pre-Exposure Prophylaxis and Antiretroviral Resistance: HIV Prevention at a Cost?

Pre-exposure prophylaxis (PrEP), the use of antiretrovirals (ARVs) by human immunodeficiency virus (HIV)–uninfected individuals to prevent acquisition of the virus during high-risk sexual encounters, enjoyed its first 2 major successes with the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 and the Pre-Exposure Prophylaxis Initiative (iPrEx). These successes were buoyed by additional positive results from the TDF2 and Partners PrEP trials. Although no seroconverters in either arm of CAPRISA developed resistance to tenofovir, 2 participants in iPrEx with undetected, seronegative acute HIV infection were randomized to receive daily oral tenofovir-emtricitabine and resistance to emtricitabine was later discovered in both men. A similar case in the TDF2 study resulted in resistance to both ARVs. These cases prompted us to examine existing literature on the nature of resistance mutations elicited by ARVs used for PrEP. Here, we discuss the impact of signature mutations selected by PrEP, how rapidly these emerge with daily ARV exposure, and the individual-level and public health consequences of ARV resistance

Spatial Epidemiology of Recently Acquired HIV Infections across Rural and Urban Areas of North Carolina

Transmission of HIV continues in the United States (US), despite prevention efforts aimed at education and treatment. Concurrently, drug resistance in HIV, particularly in patients being infected with HIV for the first time, poses a threat to the continued success of treatment for HIV positive individuals. In North Carolina, nearly one in five individuals with acute HIV infection (AHI) is infected with a drug-resistant strain, a phenomenon known as transmitted drug resistance (TDR). Few studies of AHI or TDR take into account both the spatial aspects of residence at time of infection and the genetic characteristics of the viruses, and questions remain about how viruses are transmitted across space and the rural-urban divide. Using AHI strains from North Carolina, we examined whether differences exist in the spatial patterns of AHI versus AHI with TDR, as well as whether the genetic characteristics of these HIV infections vary by rural-urban status and across Health Service Areas. The highest amounts of TDR were detected in persons under age 30, African Americans, and men who have sex with men (MSM) - similar to the populations where the highest numbers of AHI without TDR are observed. Nearly a quarter of patients reside in rural areas, and there are no significant differences between rural and urban residence among individuals infected with drug resistant or drug susceptible viruses. We observe similar levels of genetic distance between HIV found in rural and urban areas, indicating that viruses are shared across the rural-urban divide. Genetic differences are observed, however, across Health Service Areas, suggesting that local areas are sites of genetic differentiation in viruses being transmitted to newly infected individuals. These results indicate that future efforts to prevent HIV transmission need to be spatially targeted, focusing on local-level transmission in risky populations, in addition to statewide anti- HIV efforts

Human Immunodeficiency Virus Testing Practices and Interest in Self-Testing Options Among Young, Black Men Who Have Sex With Men in North Carolina

Young, Black men who have sex with men (YBMSM) experience disproportionately high HIV incidence in the United States. Relative to other at-risk populations, less is known about their HIV testing behaviors and preferences regarding self-testing

Hepatic safety and tolerability of raltegravir among HIV patients coinfected with hepatitis B and/or C

Potential liver toxicity is an important consideration for antiretroviral selection among patients coinfected with HIV and viral hepatitis (B and/or C). We sought to describe the hepatic safety profile of raltegravir in this population

Efforts at the Frontlines: Implementing a Hepatitis C Testing and Linkage-to-Care Program at the Local Public Health Level

The national Viral Hepatitis Action Plan recommends strengthening partnerships among health departments, community-based organizations, and health-care providers for hepatitis services. We implemented a hepatitis C virus (HCV) testing and linkage-to-care program through a local health department using similar strategies reported for HIV care

Opportunities for sexual transmission of antiretroviral drug resistance among HIV-infected patients in care

OBJECTIVE: To assess opportunities for transmitted drug resistance (TDR), we examined sexual risk behaviours, HIV viraemia and antiretroviral resistance among patients in care. DESIGN: A retrospective, cross-sectional analysis of clinical cohort data. METHODS: For 244 UNC Center for AIDS Research HIV Clinical Cohort participants, demographic and behavioural data were obtained during in-person interviews between 2000 and 2011. Genotypic resistance tests were interpreted using WHO surveillance drug resistance mutations (SDRMs). Log-linear binomial regression was used to evaluate associations with TDR risk, defined as unprotected sex in the prior 6 months, HIV RNA at least 400 copies/ml and at least one SDRM. RESULTS: Participants included 91 (37%) women and 153 men, of whom 92 (60%) were MSM. Median age was 43 years; 70% were Black (n = 171). Most (97%) were antiretroviral-experienced; 44% had exposure to more than four regimens. Among 204 individuals on antiretrovirals, 42% reported suboptimal adherence and 29% were viraemic. Over half of participants had at least one SDRM (n = 131); 26 (11%) had triple-class resistance. Overall, 70% were sexually active, and 55% used condoms inconsistently. Thirty (12%) reported unprotected sex during periods of drug-resistant viraemia. Higher TDR risk was associated with prior homelessness [adjusted prevalence ratio (aPR) 2.20, 95% confidence interval (CI) 1.16-4.18], active substance use (aPR 3.12, 95% CI 1.47-6.62) and nonsignificantly with MSM (aPR 1.75, 95% CI 0.93-3.28). CONCLUSION: A small but significant proportion of clinic patients with drug-resistant HIV engage in sexual behaviours that place others at risk for TDR. Targeted efforts in secondary prevention could have an impact on TDR incidence, over time

HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN