Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

<p>Abstract</p> <p>Background</p> <p>The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with <it>nef</it>-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1). Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of <it>nef</it>/long-terminal repeat (LTR) sequences demonstrated convergent <it>nef</it>/LTR sequence evolution in SBBC SP and LTNP. Thus, the <it>in vivo </it>pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than <it>nef</it>/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 <it>rev </it>alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36.</p> <p>Results</p> <p>The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1_NL4-3, C18 or C98. D36 Rev also had a 50–60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus.</p> <p>Conclusion</p> <p>These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated <it>rev </it>alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members.</p

Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues

<p>Abstract</p> <p>Background</p> <p>CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81).</p> <p>Findings</p> <p>Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop.</p> <p>Conclusions</p> <p>Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.</p

Macrophage Tropism and Cytopathicity of HIV-1 Variants Isolated Sequentially from a Long-Term Survivor Infected with nef-Deleted Virus

Long-term survival of human immunodeficiency virus type 1 (HIV-1) infection has been noted in rare cohorts of individuals infected with nef-deleted virus. Enhanced macrophage tropism and cytopathicity contribute to pathogenicity of wild type HIV-1. To better understand the pathogenesis of nef-deleted HIV-1, we analyzed the replication capacity and macrophage cytopathicity of nef-deleted HIV-1 isolated sequentially from a long-term survivor during progression to AIDS (n=6 isolates). Compared with controls, all nef-deleted viruses replicated to low levels in peripheral blood mononu-clear cells and monocyte-derived macrophages (MDM). One nef-deleted virus that was isolated on the development of AIDS caused high levels of syncytia in MDM similar to control viruses, but five viruses isolated from earlier times prior to AIDS onset caused only minimal cytopathicity. Together, these results suggest that enhanced cytopathicity of nef-deleted HIV-1 for MDM can occur independently of replication capacity, and may contribute to the pathogenesis of nef-deleted HIV-1 infection

Does publishing during the doctorate influence completion time? A quantitative study of doctoral candidates in Australia.

Aim/Purpose: This paper investigates the association between publishing during doctoral candidature and completion time. The effects of discipline and of gaining additional support through a doctoral cohort program are also explored. Background: Candidates recognize the value of building a publication track record to improve their career prospects yet are cognizant of the time it takes to publish peer-reviewed articles. In some institutions or disciplines, there is a policy or the expectation that doctoral students will publish during their candidature. However, doctoral candidates are also under increasing pressure to complete their studies within a designated timeframe. Thus, some candidates and faculty perceive the two requirements – to publish and to complete on time – as mutually exclusive. Furthermore, where candidates have a choice in the format that the PhD submission will take, be it by monograph, PhD-by-publication, or a hybrid thesis, there is little empirical evidence available to guide the decision. This paper provides a quantitative analysis of the association between publishing during candidature and time-to-degree and investigates other variables associated with doctoral candidate research productivity and efficiency. Methodology: Multivariate logistic regression analyses were used to examine the predictors (discipline [field of research], gender, age group, domestic or international student status, and belonging to a cohort program) of doctoral candidate research productivity and efficacy. Research productivity was quantified by the number of peer-reviewed journal articles that a candidate published as a primary author during and up to 24 months after thesis submission. Efficacy (time-to-degree) was quantified by the number of Full-Time Equivalent (FTE) years of candidature. Data on 1,143 doctoral graduates were obtained from a single Australian university for the period extending from 2000 to 2020. Complete publication data were available on 707 graduates, and time-to-degree data on 664 graduates. Data were drawn from eight fields of research, which were grouped into the disciplines of health, biological sciences, agricultural and environmental sciences, and chemical, earth, and physical sciences. Contribution: This paper addresses a gap in empirical literature by providing evidence of the association between publishing during doctoral candidature and time-to-degree in the disciplines of health, biological sciences, agricultural and environmental sciences, and chemical, earth, and physical sciences. The paper also adds to the body of evidence that demonstrates the value of belonging to a cohort pro-gram for doctoral student outcomes. Findings: There is a significant association between the number of articles published and median time-to-degree. Graduates with the highest research productivity (four or more articles) exhibited the shortest time-to-degree. There was also a significant association between discipline and the number of publications published during candidature. Gaining additional peer and research-focused support and training through a cohort program was also associated with higher research productivity and efficiency compared to candidates in the same discipline but not in receipt of the additional support. Recommendations for Practitioners: While the encouragement of candidates to both publish and complete within the recommended doctorate timeframe is recommended, even within disciplines characterized by high levels of research productivity, i.e., where publishing during candidature is the "norm," the desired levels of student research productivity and efficiency are only likely to be achieved where candidates are provided with consistent writing and publication-focused training, together with peer or mentor support. Recommendations for Researchers: Publishing peer-reviewed articles during doctoral candidature is shown not to adversely affect candidates' completion time. Researchers should seek writing and publication-focused support to enhance their research productivity and efficiency. Impact on Society: Researchers have an obligation to disseminate their findings for the benefit of society, industry, or practice. Thus, doctoral candidates need to be encouraged and supported to publish as they progress through their candidature. Future Research: The quantitative findings need to be followed up with a mixed-methods study aimed at identifying which elements of publication and research-focused sup-port are most effective in raising doctoral candidate productivity and efficacy

Lessons learned while searching for meaning in doctoral completion metrics: An intra-institution case study

For institutions intent on improving their research student outcomes, it is important to identify the variables most strongly associated with timely or tardy completions, which the university has the potential to influence or amend. For this to occur the analyses of doctoral completion times need to be conducted at an institution or discipline level. However, conducting meaningful analyses of doctoral completion data is a complex undertaking fraught with potential problems. This paper uses an intra-institution case study to reflect on and illustrate how different meanings can emerge from data analysis, depending on the statistical approach used to analyse the data. The paper outlines the lessons learned during data analysis, and the implications for doctoral education. The findings also point to potential marketing opportunities for research institutions

Lipid metabolism in patients infected with Nef-deficient HIV-1 strain

Background: HIV protein Nef plays a key role in impairing cholesterol metabolism in both HIV infected and bystander cells. The existence of a small cohort of patients infected with Nef-deficient strain of HIV presented a unique opportunity to test the effect of Nef on lipid metabolism in a clinical setting. Methods: Here we report the results of a study comparing six patients infected with Nef-deficient strain of HIV (Delta NefHIV) with six treatment-naive patients infected with wild-type HIV (WT HIV). Lipoprotein profile, size and functionality of high density lipoprotein (HDL) particles as well as lipidomic and microRNA profiles of patient plasma were analyzed. Results: We found that patients infected with Delta NefHIV had lower proportion of subjects with plasma HDL-C levels < 1 mmol/l compared to patients infected with WT HIV. Furthermore, compared to a reference group of HIV-negative subjects, there was higher abundance of smaller under-lipidated HDL particles in plasma of patients infected with WT HIV, but not in those infected with Delta NefHIV. Lipidomic analysis of plasma revealed differences in abundance of phosphatidylserine and sphingolipids between patients infected with Delta NefHIV and WT HIV. MicroRNA profiling revealed that plasma abundance of 24 miRNAs, many of those involved in regulation of lipid metabolism, was differentially regulated by WT HIV and Delta NefHIV. Conclusion: Our findings are consistent with HIV protein Nef playing a significant role in pathogenesis of lipid-related metabolic complications of HIV disease

Lipid metabolism in patients infected with Nef-deficient HIV-1 strain.

BACKGROUND: HIV protein Nef plays a key role in impairing cholesterol metabolism in both HIV infected and bystander cells. The existence of a small cohort of patients infected with Nef-deficient strain of HIV presented a unique opportunity to test the effect of Nef on lipid metabolism in a clinical setting. METHODS: Here we report the results of a study comparing six patients infected with Nef-deficient strain of HIV (ΔNefHIV) with six treatment-naïve patients infected with wild-type HIV (WT HIV). Lipoprotein profile, size and functionality of high density lipoprotein (HDL) particles as well as lipidomic and microRNA profiles of patient plasma were analyzed. RESULTS: We found that patients infected with ΔNefHIV had lower proportion of subjects with plasma HDL-C levels/l compared to patients infected with WT HIV. Furthermore, compared to a reference group of HIV-negative subjects, there was higher abundance of smaller under-lipidated HDL particles in plasma of patients infected with WT HIV, but not in those infected with ΔNefHIV. Lipidomic analysis of plasma revealed differences in abundance of phosphatidylserine and sphingolipids between patients infected with ΔNefHIV and WT HIV. MicroRNA profiling revealed that plasma abundance of 24 miRNAs, many of those involved in regulation of lipid metabolism, was differentially regulated by WT HIV and ΔNefHIV. CONCLUSION: Our findings are consistent with HIV protein Nef playing a significant role in pathogenesis of lipid-related metabolic complications of HIV disease

Evolution of DC-SIGN use revealed by fitness studies of R5 HIV-1 variants emerging during AIDS progression

<p>Abstract</p> <p>Background</p> <p>At early stages of infection CCR5 is the predominant HIV-1 coreceptor, but in approximately 50% of those infected CXCR4-using viruses emerge with disease progression. This coreceptor switch is correlated with an accelerated progression. However, those that maintain virus exclusively restricted to CCR5 (R5) also develop AIDS. We have previously reported that R5 variants in these "non-switch virus" patients evolve during disease progression towards a more replicative phenotype exhibiting altered CCR5 coreceptor interactions. DC-SIGN is a C-type lectin expressed by dendritic cells that HIV-1 may bind and utilize for enhanced infection of T cells in <it>trans</it>. To further explore the evolution of the R5 phenotype we analyzed sequential R5 isolates obtained before and after AIDS onset, i.e. at the chronic stage and during end-stage disease, with regard to efficiency of DC-SIGN use in <it>trans</it>-infections.</p> <p>Results</p> <p>Results from binding and <it>trans</it>-infection assays showed that R5 viruses emerging during end-stage AIDS disease displayed reduced ability to use DC-SIGN. To better understand viral determinants underlying altered DC-SIGN usage by R5 viruses, we cloned and sequenced the HIV-1 <it>env </it>gene. We found that end-stage R5 viruses lacked potential N-linked glycosylation sites (PNGS) in the gp120 V2 and V4 regions, which were present in the majority of the chronic stage R5 variants. One of these sites, amino acid position 160 (aa160) in the V2 region, also correlated with efficient use of DC-SIGN for binding and <it>trans</it>-infections. In fitness assays, where head-to-head competitions between chronic stage and AIDS R5 viruses were setup in parallel direct and DC-SIGN-mediated infections, results were further supported. Competitions revealed that R5 viruses obtained before AIDS onset, containing the V2 PNGS at aa160, were selected for in the <it>trans</it>-infection. Whereas, in agreement with our previous studies, the opposite was seen in direct target cell infections where end-stage viruses out-competed the chronic stage viruses.</p> <p>Conclusion</p> <p>Results of our study suggest R5 virus variants with diverse fitness for direct and DC-SIGN-mediated <it>trans</it>-infections evolve within infected individuals at end-stage disease. In addition, our results point to the importance of a glycosylation site within the gp120 V2 region for efficient DC-SIGN use of HIV-1 R5 viruses.</p

HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry

<p>Abstract</p> <p>Background</p> <p>Maraviroc (MVC) and other CCR5 antagonists are HIV-1 entry inhibitors that bind to- and alter the conformation of CCR5, such that CCR5 is no longer recognized by the viral gp120 envelope (Env) glycoproteins. Resistance to CCR5 antagonists results from HIV-1 Env acquiring the ability to utilize the drug-bound conformation of CCR5. Selecting for HIV-1 resistance to CCR5-antagonists <it>in vitro </it>is relatively difficult. However, the CCR5-using CC1/85 strain appears to be uniquely predisposed to acquiring resistance to several CCR5 antagonists <it>in vitro </it>including MVC, vicriviroc and AD101.</p> <p>Findings</p> <p>Here, we show that Env derived from the parental CC1/85 strain is inherently capable of a low affinity interaction with MVC-bound CCR5. However, this phenotype was only revealed in 293-Affinofile cells and NP2-CD4/CCR5 cells that express very high levels of CCR5, and was masked in TZM-bl, JC53 and U87-CD4/CCR5 cells as well as PBMC, which express comparatively lower levels of CCR5 and which are more commonly used to detect resistance to CCR5 antagonists.</p> <p>Conclusions</p> <p>Env derived from the CC1/85 strain of HIV-1 is inherently capable of a low-affinity interaction with MVC-bound CCR5, which helps explain the relative ease in which CC1/85 can acquire resistance to CCR5 antagonists <it>in vitro</it>. The detection of similar phenotypes in patients may identify those who could be at higher risk of virological failure on MVC.</p

Phenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source

<p>Abstract</p> <p>Background</p> <p>The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with attenuated, <it>nef</it>-deleted variants of human immunodeficiency virus type 1 (HIV-1) acquired from a single source. Long-term prospective studies have demonstrated that the SBBC now comprises slow progressors (SP) as well as long-term nonprogressors (LTNP). Convergent evolution of <it>nef </it>sequences in SBBC SP and LTNP indicates the <it>in vivo </it>pathogenicity of HIV-1 in SBBC members is dictated by factors other than <it>nef</it>. To better understand mechanisms underlying the pathogenicity of <it>nef</it>-deleted HIV-1, we examined the phenotype and <it>env </it>sequence diversity of sequentially isolated viruses (n = 2) from 3 SBBC members.</p> <p>Results</p> <p>The viruses characterized here were isolated from two SP spanning a three or six year period during progressive HIV-1 infection (subjects D36 and C98, respectively) and from a LTNP spanning a two year period during asymptomatic, nonprogressive infection (subject C18). Both isolates from D36 were R5X4 phenotype and, compared to control HIV-1 strains, replicated to low levels in peripheral blood mononuclear cells (PBMC). In contrast, both isolates from C98 and C18 were CCR5-restricted. Both viruses isolated from C98 replicated to barely detectable levels in PBMC, whereas both viruses isolated from C18 replicated to low levels, similar to those isolated from D36. Analysis of <it>env </it>by V1V2 and V3 heteroduplex tracking assay, V1V2 length polymorphisms, sequencing and phylogenetic analysis showed distinct intra- and inter-patient <it>env </it>evolution.</p> <p>Conclusion</p> <p>Independent evolution of <it>env </it>despite convergent evolution of <it>nef </it>may contribute to the <it>in vivo </it>pathogenicity of <it>nef</it>-deleted HIV-1 in SBBC members, which may not necessarily be associated with changes in replication capacity or viral coreceptor specificity.</p