scholarly journals A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

10.3791/51506 ◽  
2014 ◽  
Author(s):  
Daniel T. Claiborne ◽  
Jessica L. Prince ◽  
Eric Hunter
Keyword(s):  
Restriction Enzyme ◽  
Replication Capacity ◽  
Subtype C ◽  
Cloning Method ◽  
Hiv 1 ◽  
Chimeric Viruses

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility

2019 ◽  
Vol 476 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Alison Williams ◽  
Adriaan Basson ◽  
Ikechukwu Achilonu ◽  
Heini W. Dirr ◽  
Lynn Morris ◽  
...  
Keyword(s):  
Specific Activity ◽  
Catalytic Efficiency ◽  
Drug Susceptibility ◽  
Hiv Protease ◽  
Titration Calorimetry ◽  
Replication Capacity ◽  
Subtype C ◽  
Viral Replication Capacity ◽  
Hiv 1

AbstractHIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat. The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Drug susceptibility and replication capacity of a rare HIV-1 subtype C protease hinge region variant

2019 ◽  
Vol 24 (5) ◽  
pp. 333-342
Author(s):  
Jake Zondagh ◽  
Adriaan E Basson ◽  
Ikechukwu Achilonu ◽  
Lynn Morris ◽  
Heini W Dirr ◽  
...  
Keyword(s):  
Drug Susceptibility ◽  
Hinge Region ◽  
Replication Capacity ◽  
Subtype C ◽  
Hiv 1

scholarly journals A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
Cristina Sánchez-Corzo ◽  
Carlos Sorzano ◽  
Lidia Saiz ◽  
Pilar Mediavilla ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Hiv Vaccine ◽  
Replication Capacity ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara ◽  
Clade C ◽  
Membrane Bound ◽  
Hiv 1

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

scholarly journals The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0161596
Author(s):  
Eveline Santos da Silva ◽  
Martin Mulinge ◽  
Morgane Lemaire ◽  
Cécile Masquelier ◽  
Cyprien Beraud ◽  
...  
Keyword(s):  
Cytoplasmic Tail ◽  
Replication Capacity ◽  
Viral Infectivity ◽  
Subtype C ◽  
Cell Transmission ◽  
Hiv 1

scholarly journals Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Katherine A. Sutherland ◽  
Dami A. Collier ◽  
Daniel T. Claiborne ◽  
Jessica L. Prince ◽  
Martin J. Deymier ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Subtype C ◽  
Replication Efficiency ◽  
In Vitro Replication ◽  
Hiv 1

scholarly journals Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Manickam Ashokkumar ◽  
Shambhu G. Aralaguppe ◽  
Srikanth P. Tripathy ◽  
Luke Elizabeth Hanna ◽  
Ujjwal Neogi
Keyword(s):  
Hiv Infection ◽  
Biological Properties ◽  
Subtype C ◽  
Chronic Stage ◽  
Human Host ◽  
Molecular Features ◽  
Successful Infection ◽  
Hiv Research ◽  
Hiv 1 ◽  
Chimeric Viruses

ABSTRACT Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection. IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

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 Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

2010 ◽  
Vol 84 (20) ◽  
pp. 10820-10831 ◽  
Author(s):  
Jaclyn K. Wright ◽  
Zabrina L. Brumme ◽  
Jonathan M. Carlson ◽  
David Heckerman ◽  
Carl M. Kadie ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Population Level ◽  
Hla Class I ◽  
Viral Fitness ◽  
Baseline Viral Load ◽  
Replication Capacity ◽  
Subtype C ◽  
Fitness Costs ◽  
The Impact ◽  
Hiv 1

ABSTRACT The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

scholarly journals Linkages between HIV-1 specificity for CCR5 or CXCR4 and in vitro usage of alternative coreceptors during progressive HIV-1 subtype C infection

Retrovirology ◽  
2013 ◽  
Vol 10 (1) ◽  
pp. 98 ◽  
Author(s):  
Kieran Cashin ◽  
Martin R Jakobsen ◽  
Jasminka Sterjovski ◽  
Michael Roche ◽  
Anne Ellett ◽  
...  
Keyword(s):  
Subtype C ◽  
Hiv 1
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