scholarly journals Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Marion W. Kiguoya ◽  
Jaclyn K. Mann ◽  
Denis Chopera ◽  
Kamini Gounder ◽  
Guinevere Q. Lee ◽  
...  
Keyword(s):  
Disease Progression ◽  
Sub Saharan Africa ◽  
Replication Capacity ◽  
Binding Motif ◽  
Replicative Capacity ◽  
Subtype C ◽  
East African ◽  
Recombinant Viruses ◽  
Subtype B ◽  
Hiv 1

ABSTRACT There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates (r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases (P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants (P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that HIV-1 Gag-protease-driven replication capacity correlates with the replication capacity of whole virus isolates. We further show that subtype B displays a significantly higher Gag-protease-mediated replication capacity than does subtype C, and we identify a major genetic determinant of these differences. Moreover, in two independent East African cohorts we demonstrate a reproducible hierarchy of Gag-protease-driven replicative capacity, whereby recombinants exhibit the greatest replication, followed by subtype D, followed by subtypes A and C. Our data identify Gag-protease as a major determinant of subtype differences in disease progression among HIV-1 subtypes; furthermore, we propose that the poorer viral replicative capacity of subtypes A and C may paradoxically contribute to their more efficient spread in sub-Saharan Africa.

scholarly journals Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness

2012 ◽  
Vol 93 (12) ◽  
pp. 2625-2634 ◽  
Author(s):  
Elena Capel ◽  
Glòria Martrus ◽  
Mariona Parera ◽  
Bonaventura Clotet ◽  
Miguel Angel Martínez
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Replication Capacity ◽  
Recombinant Viruses ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

scholarly journals Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C

2011 ◽  
Vol 85 (8) ◽  
pp. 3996-4006 ◽  
Author(s):  
J. K. Wright ◽  
V. Novitsky ◽  
M. A. Brockman ◽  
Z. L. Brumme ◽  
C. J. Brumme ◽  
...  
Keyword(s):  
Disease Progression ◽  
Replication Capacity ◽  
Subtype C ◽  
Hiv 1

scholarly journals Construction and validation of a plasmid for HIV-1 phenotyping assays using Luminescence

2021 ◽  
Vol 13 (4) ◽  
pp. e7209
Author(s):  
Bianca Cristina Duarte Vivarini ◽  
Aislan de Carvalho Vivarini
Keyword(s):  
Light Emission ◽  
Luciferase Reporter ◽  
Integrase Inhibitors ◽  
Subtype C ◽  
Luciferase Reporter Gene ◽  
Integrase Gene ◽  
Recombinant Viruses ◽  
Subtype B ◽  
Viral Particles ◽  
Hiv 1

Objective: The objective of these studies was the construction of a recombination vector for HIV-1 Integrase containing luciferase reporter gene, for the generation of recombinant viruses that can be used in phenotyping assays with integrase inhibitors. Methods: In this work, the vector pNL4-3Luc was molecularly manipulated in order to delete the integrase gene and build a vector for recombination of integrases from different virus subtypes. Results: As a result, viruses with recombinant integrases were generated in HEK293T cells transfected with plasmids, showing significant levels of luminescence. After the purification of the produced viral particles, susceptible lymphocytes were infected with the viruses containing the recombinant integrases and luminescence was detected in both integrases from HIV-1 subtype B and subtype C. Conclusion: The observation of light emission from cells infected by viruses with different integrases can be an efficient method to assess the susceptibility of these viruses in the presence of specific inhibitors for HIV-1 Integrase.

scholarly journals Impact of the N348I Mutation in HIV-1 Reverse Transcriptase on Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Non-Subtype B HIV-1

2011 ◽  
Vol 55 (4) ◽  
pp. 1806-1809 ◽  
Author(s):  
Adele L. McCormick ◽  
Chris M. Parry ◽  
Anne Crombe ◽  
Ruth L. Goodall ◽  
Ravindra K. Gupta ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Drug Susceptibility ◽  
Replication Capacity ◽  
Replicative Capacity ◽  
Double Mutation ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
Subtype B ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACTWe investigated the effect of N348I alone and with M184V on nonnucleoside reverse transcriptase inhibitor (NNRTI) drug susceptibility and replicative capacity in B and non-B HIV-1 isolates. N348I reduced the susceptibility to all NNRTI drugs across subtypes. The replication capacity of all viruses in a variety of cell lines was impaired by N348I. Interestingly, the N348I and M184V double mutation compensated for the reduced NNRTI drug susceptibility observed in the N348I single mutant and marginally improved viral replicative capacity.

scholarly journals HIV-1 Protease Codon 36 Polymorphisms and Differential Development of Resistance to Nelfinavir, Lopinavir, and Atazanavir in Different HIV-1 Subtypes

2010 ◽  
Vol 54 (7) ◽  
pp. 2878-2885 ◽  
Author(s):  
Irene Lisovsky ◽  
Susan M. Schader ◽  
Jorge-Luis Martinez-Cajas ◽  
Maureen Oliveira ◽  
Daniela Moisi ◽  
...  
Keyword(s):  
Viral Replication ◽  
Resistance Mutation ◽  
Fold Increase ◽  
Replication Capacity ◽  
Protease Gene ◽  
Subtype C ◽  
Phenotypic Analysis ◽  
Subtype B ◽  
Viral Replication Capacity ◽  
Hiv 1

ABSTRACT The amino acid at position 36 of the HIV-1 protease differs among various viral subtypes, in that methionine is usually found in subtype B viruses but isoleucine is common in other subtypes. This polymorphism is associated with higher rates of treatment failure involving protease inhibitors (PIs) in non-subtype B-infected patients. To investigate this, we generated genetically homogeneous wild-type viruses from subtype B, subtype C, and CRF02_AG full-length molecular clones and showed that subtype C and CRF02_AG I36 viruses exhibited higher levels of resistance to various PIs than their respective M36 counterparts, while the opposite was observed for subtype B viruses. Selections for resistance with each variant were performed with nelfinavir (NFV), lopinavir (LPV), and atazanavir (ATV). Sequence analysis of the protease gene at week 35 revealed that the major NFV resistance mutation D30N emerged in NFV-selected subtype B viruses and in I36 subtype C viruses, despite polymorphic variation. A unique mutational pattern developed in subtype C M36 viruses selected with NFV or ATV. The presence of I47A in LPV-selected I36 CRF02_AG virus conferred higher-level resistance than L76V in LPV-selected M36 CRF02_AG virus. Phenotypic analysis revealed a >1,000-fold increase in NFV resistance in I36 subtype C NFV-selected virus with no apparent impact on viral replication capacity. Thus, the position 36 polymorphism in the HIV-1 protease appears to have a differential effect on both drug susceptibility and the viral replication capacity, depending on both the viral subtype and the drug being evaluated.

scholarly journals Pol-Driven Replicative Capacity Impacts Disease Progression in HIV-1 Subtype C Infection

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Doty B. A. Ojwach ◽  
Daniel MacMillan ◽  
Tarylee Reddy ◽  
Vladimir Novitsky ◽  
Zabrina L. Brumme ◽  
...  
Keyword(s):  
Viral Load ◽  
T Cell ◽  
Disease Progression ◽  
Chronic Infection ◽  
Hiv Disease ◽  
Subtype C ◽  
Recombinant Viruses ◽  
Immune Mediated ◽  
Hiv 1

ABSTRACT CD8+ T cell-mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a green fluorescent protein (GFP) reporter T cell assay. In recently infected individuals, reverse transcriptase (RT)-integrase-driven RC correlated significantly with viral load set point (r = 0.25; P = 0.03) and CD4+ T cell decline (P = 0.013). Moreover, significant associations between RT integrase-driven RC and viral load (r = 0.28; P < 0.0001) and CD4+ T cell count (r = −0.29; P < 0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (P = 0.05), as was expression of HLA-B*07 (P = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (P = 0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K, and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally described HLA-restricted cytotoxic T-lymphocyte epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC. IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein, but the impact of Pol function on HIV disease progression is not well defined. Here, we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C-infected individuals with recent and chronic infection and measured their ability to replicate in vitro. We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.

Non-B HIV-1 subtypes in sub-Saharan Africa: impact of subtype on protease inhibitor efficacy

2014 ◽  
Vol 395 (10) ◽  
pp. 1151-1161 ◽  
Author(s):  
Previn Naicker ◽  
Yasien Sayed
Keyword(s):  
South Africa ◽  
Sub Saharan Africa ◽  
Resistance Mutations ◽  
Subtype C ◽  
Subtype B ◽  
Aids Pandemic ◽  
Sub Saharan ◽  
Immunodeficiency Syndrome ◽  
Living With Hiv ◽  
Hiv 1

Abstract In 2012, 25 million people [71% of global human immunodeficiency virus (HIV) infection] were estimated to be living with HIV in sub-Saharan Africa. Of these, approximately 1.6 million were new infections and 1.2 million deaths occurred. South Africa alone accounted for 31% of HIV/acquired immunodeficiency syndrome (AIDS) deaths in sub-Saharan Africa. This disturbing statistic indicates that South Africa remains the epicenter of the HIV/AIDS pandemic, compounded by the fact that only 36% of HIV-positive patients in South Africa have access to antiretroviral (ARV) treatment. Drug resistance mutations have emerged, and current ARVs show reduced efficacy against non-B subtypes. In addition, several recent studies have shown an increased prevalence of non-B African HIV strains in the Americas and Europe. Therefore, the use of ARVs in a non-B HIV-1 subtype context requires further investigation. HIV-1 subtype C protease, found largely in sub-Saharan Africa, has been under-investigated when compared with the subtype B protease, which predominates in North America and Europe. This review, therefore, focuses on HIV-1 proteases from B and C subtypes.

scholarly journals Correction for Ojwach et al., “Pol-Driven Replicative Capacity Impacts Disease Progression in HIV-1 Subtype C Infection”

2019 ◽  
Vol 93 (3) ◽  
Author(s):  
Doty B. A. Ojwach ◽  
Daniel MacMillan ◽  
Tarylee Reddy ◽  
Vladimir Novitsky ◽  
Zabrina L. Brumme ◽  
...  
Keyword(s):  
Disease Progression ◽  
Replicative Capacity ◽  
Subtype C ◽  
Hiv 1

scholarly journals Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

2010 ◽  
Vol 54 (9) ◽  
pp. 3938-3948 ◽  
Author(s):  
Francesca Ceccherini-Silberstein ◽  
Kurt Van Baelen ◽  
Daniele Armenia ◽  
Maria Trignetti ◽  
Evelien Rondelez ◽  
...  
Keyword(s):  
Highly Active Antiretroviral Therapy ◽  
Novel Mutation ◽  
Rnase H ◽  
Replication Capacity ◽  
Transmitted Drug Resistance ◽  
Resistance Mutations ◽  
Primary Resistance ◽  
Recombinant Viruses ◽  
Subtype B ◽  
Hiv 1

ABSTRACTThe goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate theirin vitroeffects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

scholarly journals Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Vanessa L. Naidoo ◽  
Jaclyn K. Mann ◽  
Christie Noble ◽  
Emily Adland ◽  
Jonathan M. Carlson ◽  
...  
Keyword(s):  
Viral Replication ◽  
Replication Capacity ◽  
Mother To Child Transmission ◽  
Subtype C ◽  
Child Transmission ◽  
Recombinant Viruses ◽  
Viral Replication Capacity ◽  
Viral Determinants ◽  
Mother To Child ◽  
Hiv 1

ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that “consensus-like” virus sequences correspond to lower in vitro replication abilities yet appear to be preferentially transmitted suggests that viral characteristics favoring transmission are decoupled from those that enhance replicative capacity.

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