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

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.

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

Journal of Virology ◽

10.1128/jvi.00253-17 ◽

2017 ◽

Vol 91 (13) ◽

Cited By ~ 22

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.

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Genetic and Neutralization Properties of Subtype C Human Immunodeficiency Virus Type 1 Molecular env Clones from Acute and Early Heterosexually Acquired Infections in Southern Africa

Journal of Virology ◽

10.1128/jvi.01730-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11776-11790 ◽

Cited By ~ 276

Author(s):

Ming Li ◽

Jesus F. Salazar-Gonzalez ◽

Cynthia A. Derdeyn ◽

Lynn Morris ◽

Carolyn Williamson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralizing Antibody ◽

Luciferase Reporter ◽

Subtype C ◽

Subtype B ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

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A Single Polymorphism in HIV-1 Subtype C SP1 Is Sufficient To Confer Natural Resistance to the Maturation Inhibitor Bevirimat

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01435-10 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3324-3329 ◽

Cited By ~ 29

Author(s):

Wuxun Lu ◽

Karl Salzwedel ◽

Dan Wang ◽

Suvobrata Chakravarty ◽

Eric O. Freed ◽

...

Keyword(s):

Natural Resistance ◽

Small Scale ◽

Amino Acid Residues ◽

Subtype C ◽

Gag Protein ◽

Subtype B ◽

Viral Particles ◽

Maturation Inhibitor ◽

Primary Determinant ◽

Hiv 1

ABSTRACT3-O-(3′,3′-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.

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Nationwide Study of Drug Resistance Mutations in HIV-1 Infected Individuals under Antiretroviral Therapy in Brazil

International Journal of Molecular Sciences ◽

10.3390/ijms22105304 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5304

Author(s):

Ana Santos-Pereira ◽

Vera Triunfante ◽

Pedro M. M. Araújo ◽

Joana Martins ◽

Helena Soares ◽

...

Keyword(s):

Drug Resistance ◽

People Living With Hiv ◽

Resistance Mutations ◽

Subtype C ◽

Viral Loads ◽

Drug Resistance Mutations ◽

Subtype B ◽

Low Prevalence ◽

Living With Hiv ◽

Hiv 1

The success of antiretroviral treatment (ART) is threatened by the emergence of drug resistance mutations (DRM). Since Brazil presents the largest number of people living with HIV (PLWH) in South America we aimed at understanding the dynamics of DRM in this country. We analyzed a total of 20,226 HIV-1 sequences collected from PLWH undergoing ART between 2008–2017. Results show a mild decline of DRM over the years but an increase of the K65R reverse transcriptase mutation from 2.23% to 12.11%. This increase gradually occurred following alterations in the ART regimens replacing zidovudine (AZT) with tenofovir (TDF). PLWH harboring the K65R had significantly higher viral loads than those without this mutation (p < 0.001). Among the two most prevalent HIV-1 subtypes (B and C) there was a significant (p < 0.001) association of K65R with subtype C (11.26%) when compared with subtype B (9.27%). Nonetheless, evidence for K65R transmission in Brazil was found both for C and B subtypes. Additionally, artificial neural network-based immunoinformatic predictions suggest that K65R could enhance viral recognition by HLA-B27 that has relatively low prevalence in the Brazilian population. Overall, the results suggest that tenofovir-based regimens need to be carefully monitored particularly in settings with subtype C and specific HLA profiles.

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Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3456-3461.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3456-3461 ◽

Cited By ~ 22

Author(s):

Mervi Tenhami ◽

Kaisa Hakkila ◽

Matti Karp

Keyword(s):

Staphylococcus Aureus ◽

High Throughput Screening ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Light Emission ◽

Detection System ◽

Firefly Luciferase ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Luciferase Reporter Gene

ABSTRACT The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain,Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-induciblecadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

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Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

Journal of Virology ◽

10.1128/jvi.76.8.3881-3891.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3881-3891 ◽

Cited By ~ 37

Author(s):

Neerja Kaushik ◽

Amartya Basu ◽

Paul Palumbo ◽

Rene L. Myers ◽

Virendra N. Pandey

Keyword(s):

Human Immunodeficiency Virus ◽

Antiviral Treatment ◽

Significant Inhibition ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Nucleotide Analog ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.

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Genetic diversity and primary resistance among HIV-1-positive patients from Maringá, Paraná, Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652012000400005 ◽

2012 ◽

Vol 54 (4) ◽

pp. 207-213 ◽

Cited By ~ 8

Author(s):

Karine Vieira Gaspareto ◽

Flávia Myrian Martins de Almeida Mello ◽

José Ricardo Colleti Dias ◽

Vera Alice Fernandes Meneguetti ◽

Marta Evelyn Giansante Storti ◽

...

Keyword(s):

Specific Antigen ◽

Resistance Mutation ◽

Drug Resistance Mutation ◽

Subtype C ◽

Primary Resistance ◽

Subtype B ◽

Pcr Products ◽

Treatment Naïve ◽

Positive Treatment ◽

Hiv 1

The objective of this study is to identify subtypes of Human Immunodeficiency Virus type 1 (HIV-1) and to analyze the presence of mutations associated to antiretroviral resistance in the protease (PR) and reverse transcriptase (RT) regions from 48 HIV-1 positive treatment naïve patients from an outpatient clinic in Maringá, Paraná, Brazil. Sequencing was conducted using PR, partial RT and group-specific antigen gene (gag) nested PCR products from retrotranscribed RNA. Transmitted resistance was determined according to the Surveillance Drug Resistance Mutation List (SDRM) algorithm. Phylogenetic and SimPlot analysis of concatenated genetic segments classified sequences as subtype B 19/48 (39.6%), subtype C 12/48 (25%), subtype F 4/48 (8.3%), with 13/48 (27.1%) recombinant forms. Most recombinant forms were B mosaics (B/F 12.5%, B/C 10.4%), with one C/F (2.1%) and one complex B/C/F mosaic (2.1%). Low levels of transmitted resistance were found in this study, 2/48 (2.1% to NRTIs and 2.1% for PI). This preliminary data may subsidize the monitoring of the HIV evolution in the region.

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Efficiencies of Four Versions of the AMPLICOR HIV-1 MONITOR Test for Quantification of Different Subtypes of Human Immunodeficiency Virus Type 1

Journal of Clinical Microbiology ◽

10.1128/jcm.37.1.110-116.1999 ◽

1999 ◽

Vol 37 (1) ◽

pp. 110-116 ◽

Cited By ~ 74

Author(s):

K. Triques ◽

J. Coste ◽

J. L. Perret ◽

C. Segarra ◽

E. Mpoudi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Load ◽

Pcr Primers ◽

Load Test ◽

Subtype C ◽

Subtype B ◽

Immunodeficiency Virus ◽

Subtype A ◽

Hiv 1

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1.0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1.0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log10 HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (<0.5 log10). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.

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Characterization of HIV-1 Integrase Gene and Resistance Associated Mutations Prior to Roll out of Integrase Inhibitors by Kenyan National HIV-Treatment Program in Kenya

Ethiopian Journal of Health Sciences ◽

10.4314/ejhs.v30i1.6 ◽

2020 ◽

Vol 30 (1) ◽

Author(s):

Mabeya Sepha ◽

Nyamache Anthony ◽

Ngugi Caroline ◽

Nyerere Andrew ◽

Lihana Raphael

Keyword(s):

Drug Resistance ◽

Direct Sequencing ◽

Integrase Inhibitor ◽

Hiv Treatment ◽

Strand Transfer ◽

Hiv Integrase ◽

Integrase Inhibitors ◽

Resistance Mutations ◽

Integrase Gene ◽

Hiv 1

BACKGROUND: Antiretroviral therapy containing an integrase strand transfer inhibitor plus two Nucleoside Reverse Transcriptase inhibitors has now been recommended for treatment of HIV-1-infected patients. This thus determined possible pre-existing integrase resistance associated mutations in the integrase gene prior to introduction of integrase inhibitors combination therapy in Kenya.METHODS: Drug experienced HIV patients were enrolled at Kisii Teaching and Referral in Kenya. Blood specimens from (33) patients were collected for direct sequencing of HIV-1 polintegrase genes. Drug resistance mutations were interpreted according to the Stanford algorithm and phylogenetically analysed using insilico tools.RESULTS: From pooled 188 Kenyan HIV integrase sequences that were analysed for drug resistance, no major mutations conferring resistance to integrase inhibitors were detected. However, polymorphic accessory mutations associated with reduced susceptibility of integrase inhibitors were observed in low frequency; M50I (12.2%), T97A (3.7%), S153YG, E92G (1.6%), G140S/A/C (1.1%) and E157Q (0.5%). Phylogenetic analysis (330 sequences revealed that HIV-1 subtype A1 accounted for majority of the infections, 26 (78.8%), followed by D, 5 (15.2%) and C, 2 (6%).CONCLUSION: The integrase inhibitors will be effective in Kenya where HIV-1 subtype A1 is still the most predominant. However, occurring polymorphisms may warrant further investigation among drug experienced individuals on dolutegravir combination or integrase inhibitor treatment.

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