Modeling of the HIV-1 Life Cycle in Productively Infected Cells to Predict Novel Therapeutic Targets

There are many studies that model the within-host population dynamics of Human Immunodeficiency Virus Type 1 (HIV-1) infection. However, the within-infected-cell replication of HIV-1 remains to be not comprehensively addressed. There exist rather few quantitative models describing the regulation of the HIV-1 life cycle at the intracellular level. In treatment of HIV-1 infection, there remain issues related to side-effects and drug-resistance that require further search “...for new and better drugs, ideally targeting multiple independent steps in the HIV-1 replication cycle” (as highlighted recently by Tedbury & Freed, The Future of HIV-1 Therapeutics, 2015). High-resolution mathematical models of HIV-1 growth in infected cells provide an additional analytical tool in identifying novel drug targets. We formulate a high-dimensional model describing the biochemical reactions underlying the replication of HIV-1 in target cells. The model considers a nonlinear regulation of the transcription of HIV-1 mediated by Tat and the Rev-dependent transport of fully spliced and singly spliced transcripts from the nucleus to the cytoplasm. The model is calibrated using available information on the kinetics of various stages of HIV-1 replication. The sensitivity analysis of the model is performed to rank the biochemical processes of HIV-1 replication with respect to their impact on the net production of virions by one actively infected cell. The ranking of the sensitivity factors provides a quantitative basis for identifying novel targets for antiviral therapy. Our analysis suggests that HIV-1 assembly depending on Gag and Tat-Rev regulation of transcription and mRNA distribution present two most critical stages in HIV-1 replication that can be targeted to effectively control virus production. These processes are not covered by current antiretroviral treatments

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo.

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27(-) phenotype was associated with active TB in HIV(-) (p = 0.0003) and HIV(+) (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV(-) subjects, MTB-specific CD4 T cell populations from HIV(+) TB-asymptomatic subjects were often dominated by CD27(-) cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression

The molecular epidemiology of human immunodeficiency virus type 1 in six cities in Britain and Ireland

The authors sequenced the p17 coding regions of the gag gene from 211 patients infected either through injecting drug use (IDU) or by sexual intercourse between men from six cities in Scotland, N. England, N. Ireland, and the Republic of Ireland. All sequences were of subtype 5. Phylogenetic analysis revealed substantial heterogeneity in the sequences from homosexual men. In contrast, sequence from over 80% of IDUs formed a relatively tight cluster, distinct both from those of published isolates and of the gay men. There was no large-scale clustering of sequences by city in either risk group, although a number of close associations between pairs of individuals were observed. From the known date of the HIV-1 epidemic among IDUs in Edinburgh, the rate of sequence divergence at synonymous sites is estimated to be about 0.8%. On this basis it has been estimated that the date of divergence of the sequences among homosexual men to be about 1975, which may correspond to the origin of the B subtype epidemic

Multiplexed DNA Sequence Capture of Mitochondrial Genomes Using PCR Products

BACKGROUND: To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA(II) instrument. CONCLUSIONS/SIGNIFICANCE: This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA

Evolution favors protein mutational robustness in sufficiently large populations

BACKGROUND: An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. RESULTS: Here we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by existing mathematical theories, and can be quantitatively related to the way that the proteins occupy their neutral network. CONCLUSIONS: Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon may contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations

Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Performance of Ultra-Deep Pyrosequencing in Analysis of HIV-1 pol Gene Variation

INTRODUCTION: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed. PRINCIPAL FINDING: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads. CONCLUSION: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage

Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection

HIV and Mycobacterium tuberculosis (MTB) are two widespread and highly successful microbes whose synergy in pathogenesis has created a significant threat for human health globally. In acknowledgement of this fact, the European Union (EU) has funded a multinational support action, the European Network for global cooperation in the field of AIDS and TB (EUCO-Net), that brings together experts from Europe and those regions that bear the highest burden of HIV/MTB co-infection. Here, we summarise the main outcome of the EUCO-Net project derived from an expert group meeting that took place in Stellenbosch (South Africa) (AIDS/TB Workshop on Research Challenges and Opportunities for Future Collaboration) and the subsequent discussions, and propose priority areas for research and concerted actions that will have impact on future EU calls

Dual and recombinant infections: an integral part of the HIV-1 epidemic in Brazil.

We systematically evaluated multiple and recombinant infections in an HIV-infected population selected for vaccine trials. Seventy-nine HIV-1 infected persons in a clinical cohort study in Rio de Janeiro, Brazil, were evaluated for 1 year. A combination of molecular screening assays and DNA sequencing showed 3 dual infections (3.8%), 6 recombinant infections (7.6%), and 70 (88.6%) infections involving single viral subtypes. In the three dual infections, we identified HIV-1 subtypes F and B, F and D, and B and D; in contrast, the single and recombinant infections involved only HIV-1 subtypes B and F. The recombinants had five distinct B/F mosaic patterns: Bgag-p17/Bgag-p24/Fpol/Benv, Fgag-p17/Bgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Benv, and Fgag-p17/B-Fgag-p24/Fpol/Fenv. No association was found between dual or recombinant infections and demographic or clinical variables. These findings indicate that dual and recombinant infections are emerging as an integral part of the HIV/AIDS epidemic in Brazil and emphasize the heterogenous character of epidemics emerging in countries where multiple viral subtypes coexist

Low Seroprevalence of West Nile Virus in Blood Donors from Catalonia, Spain

West Nile virus (WNV) is an emerging arbovirus first recognized in Europe in the 1950s. Since then, outbreaks have been reported in several European countries. In 2010, the first WNV outbreak was recorded in Spain, affecting the southern part of the country. We conducted a seroprevalence study in the Catalonia region (northeastern Spain), an area considered at high risk of arbovirus transmission. A total of 800 serum samples from blood donors were collected and screened for antibodies against WNV by enzyme-linked immunosorbent assay (ELISA) and confirmed by a microneutralization assay. More than 50 samples tested positive by ELISA, but only one sample contained neutralizing antibodies against WNV and was obtained from a donor native of Pakistan. The low seroprevalence detected may serve as reference baseline data for monitoring WNV activity in our region in future years