Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Quasispecies dynamics and treatment outcome during early hepatitis C infection in a cohort of HIV-infected men

Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

NUTRITION AND HEALTH STATUS OF HIV-INFECTED ADULTS ON ARVS AT AMREF CLINIC KIBERA URBAN SLUM, KENYA

The health of people living with HIV and AIDS (PLWHA) is nutritionally challenged in many nations of the world. The scourge has reduced socio-economic progress globally and more so in sub-Saharan Africa (SSA) where its impact has been compounded by poverty and food insecurity. Good nutrition with proper drug use improves the quality of life for those infected but it is not known how PLWHA exposed to chronic malnutrition and food shortages from developing nations adjust their nutrition with use of Anti-Retro-viral Drugs (ARVs). This study assessed nutritional status, dietary practices, and dietary management of common illnesses that hinder daily food intake by the patients and use of ARVs with food recommendations provided by the health care givers. A descriptive case study design was used to sample 120 HIV-infected patients using systematic sampling procedure. These patients sought health care from an urban slum, Kibera AMREF clinic. Data were collected by anthropometric measurements, bio-chemical analysis, semi-structured questionnaire and secondary data. The Statistical Package for Social Sciences (SPSS) and the Nutri-Survey software packages were used to analyze data. Dietary intakes of micro-nutrients were inadequate for >70% of the patients when compared to the Recommended Daily Requirements. When Body Mass Indices (BMI) were used, only 6.7% of the respondents were underweight (BMI<18.5kg/m2) and 9.2% were overweight (BMI> 25kg/m2), serum albumin test results (mean 3.34±0.06g/dl) showed 60.8% of the respondents were protein deficient and this was confirmed by low dietary protein intakes. The BMI was not related to dietary nutrient intakes, serum albumin and CD4 cell counts (p>0.05). It appeared that there was no significant difference in BMI readings at different categories of CD4 cell count (p>0.05) suggesting that the level of immunity did not affect weight gain with ARV as observed in many studies from developed countries. Malnutrition was, therefore, evident among the 60.8% of the cases as identified by serum albumin tests and food intake was not adequate (68%) for the patients as they ate once a day due to lack of food. National food and nutrition policy should incorporate food security boosting guidelines for the poor people infected with HIV and using ARVs.

Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3′-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg2+ ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg2+ (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

Comparison of 3D finite element analysis derived stiffness and BMD to determine the failure load of the excised proximal femur

Introduction: Bone mineral density (BMD) is currently the preferred surrogate for bone strength in clinical practice. Finite element analysis (FEA) is a computer simulation technique that can predict the deformation of a structure when a load is applied, providing a measure of stiffness (Nmm−1). Finite element analysis of X-ray images (3D-FEXI) is a FEA technique whose analysis is derived froma single 2D radiographic image. Methods: 18 excised human femora had previously been quantitative computed tomography scanned, from which 2D BMD-equivalent radiographic images were derived, and mechanically tested to failure in a stance-loading configuration. A 3D proximal femur shape was generated from each 2D radiographic image and used to construct 3D-FEA models. Results: The coefficient of determination (R2%) to predict failure load was 54.5% for BMD and 80.4% for 3D-FEXI. Conclusions: This ex vivo study demonstrates that 3D-FEXI derived from a conventional 2D radiographic image has the potential to significantly increase the accuracy of failure load assessment of the proximal femur compared with that currently achieved with BMD. This approach may be readily extended to routine clinical BMD images derived by dual energy X-ray absorptiometry. Crown Copyright © 2009 Published by Elsevier Ltd on behalf of IPEM. All rights reserved

Comparison of 3D finite element analysis derived stiffness and BMD to determine the failure load of the excised proximal femur

Bone mineral density (BMD) is currently the preferred surrogate for bone strength in clinical practice. Finite element analysis (FEA) is a computer simulation technique that can predict the deformation of a structure when a load is applied, providing a measure of stiffness (N mm− 1). Finite element analysis of X-ray images (3D-FEXI) is a FEA technique whose analysis is derived from a single 2D radiographic image. This ex-vivo study demonstrates that 3D-FEXI derived from a conventional 2D radiographic image has the potential to significantly increase the accuracy of failure load assessment of the proximal femur compared with that currently achieved with BMD.

Gender inequality and the spread of HIV-AIDS in India

Purpose The purpose of the paper is to analyze the low status of women as being a major contributor for the observed gender inequality in the spread of HIV/AIDS in India. Design/methodology/approach The paper uses data from National Aids Control Organization (NACO), National Family Health Survey (NFHS 3), and the Directorate of Economics and Statistics. Findings This study highlights the problems facing women in deterring the spread of HIV/AIDS in India. The status and empowerment of women are important variables in combating the disease among both men and women in India. Literacy, education, exposure to the media, labor market participation, awareness of HIV/AIDS, and economic independence are important considerations in improving the status of women in India. Policymakers need to focus on gender inequality in order to combat the spread of HIV/AIDS in India. Originality/value While absolute figures indicate men are more likely to be infected with HIV/AIDS, the rate of decline is higher for men compared to women in India. We explore several plausible explanations for such observed inequality in the spread of HIV/AIDS across gender. In particular, a potentially important factor - the low status of women in society is attributable as an impediment to the spread of the disease. A case study of the relationship between gender empowerment and the spread of HIV/AIDS in the state with the highest concentration, Manipur, provides more insight to the difficulties faced by women in combating HIV/AIDS in India.

Behavioural beliefs concerning gender and high-risk sexual behaviours in the context of HIV/AIDS in PNG : views from within teacher education

Education is often viewed as a key approach to address sexual-health issues; the current concern is the burgeoning HIV/AIDS epidemic. This ethnographic study investigates the gender practices associated with high-risk sexual behaviour in Papua New Guinea as viewed by educators there. A number of practices, including gender inequality and associated sexual behaviours have been highlighted by male and female participants as escalating PNG’s HIV/AIDS epidemic. The study finds that although participants were well-informed concerning HIV/AIDS, they had varying beliefs concerning the prevailing gender/sexual issues involved in escalating highrisk behaviour and how to address the problem. The study further examines the behavioural beliefs and intentions of the educators themselves. Subsequently, within the data a number of underpinning factors, pertaining to gender, education and life experience, were found to be related to the behaviour beliefs and intentions of participants towards embracing change with regard to behaviours associated with gender equality in PNG. These factors appeared to encourage participants to adopt healthier gender and sexual behavioural intentions and, arguably, could provide the basis for ways to help address the gender inequality and high-risk behaviours associated with HIV/AIDS in PNG.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Background: HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. FINDINGS We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4[degree sign]C, --20[degree sign]C and -70[degree sign]C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70[degree sign]C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70[degree sign]C for 12 months is most effective in retaining VLP stability.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

Using mathematical modelling to evaluate drivers and predict trajectories of HIV and STI epidemics in South East Asian and Australian populations

Objective To evaluate the potential impact of the current global economic crisis (GEC) on the spread of HIV. Design To evaluate the impact of the economic downturn we studied two distinct HIV epidemics in Southeast Asia: the generalized epidemic in Cambodia where incidence is declining and the epidemic in Papua New Guinea (PNG) which is in an expansion phase. Methods Major HIV-related risk factors that may change due to the GEC were identified and a dynamic mathematical transmission model was developed and used to forecast HIV prevalence, diagnoses, and incidence in Cambodia and PNG over the next 3 years. Results In Cambodia, the total numbers of HIV diagnoses are not expected to be largely affected. However, an estimated increase of up to 10% in incident cases of HIV, due to potential changes in behavior, may not be observed by the surveillance system. In PNG, HIV incidence and diagnoses could be more affected by the GEC, resulting in respective increases of up to 17% and 11% over the next 3 years. Decreases in VCT and education programs are the factors that may be of greatest concern in both settings. A reduction in the rollout of antiretroviral therapy could increase the number of AIDS-related deaths (by up to 7.5% after 3 years). Conclusions The GEC is likely to have a modest impact on HIV epidemics. However, there are plausible conditions under which the economic downturns can noticeably influence epidemic trends. This study highlights the high importance of maintaining funding for HIV programs.

Serosorting may increase the risk of HIV acquisition among men who have sex with men

Background: Serosorting, the practice of seeking to engage in unprotected anal intercourse with partners of the same HIV status as oneself, has been increasing among men who have sex with men. However, the effectiveness of serosorting as a strategy to reduce HIV risk is unclear, especially since it depends on the frequency of HIV testing. Methods: We estimated the relative risk of HIV acquisition associated with serosorting compared with not serosorting by using a mathematical model, informed by detailed behavioral data from a highly studied cohort of gay men. Results: We demonstrate that serosorting is unlikely to be highly beneficial in many populations of men who have sex with men, especially where the prevalence of undiagnosed HIV infections is relatively high. We find that serosorting is only beneficial in reducing the relative risk of HIV transmission if the prevalence of undiagnosed HIV infections is less than ∼20% and ∼40%, in populations of high (70%) and low (20%) treatment rates, respectively, even though treatment reduces the absolute risk of HIV transmission. Serosorting can be expected to lead to increased risk of HIV acquisition in many settings. In settings with low HIV testing rates serosorting can more than double the risk of HIV acquisition. Conclusions: Therefore caution should be taken before endorsing the practice of serosorting. It is very important to continue promotion of frequent HIV testing and condom use, particularly among people at high risk.