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In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
- Publication Year :
- 2017
- Publisher :
- eScholarship, University of California, 2017.
-
Abstract
- The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line.
- Subjects :
- 0301 basic medicine
CD4-Positive T-Lymphocytes
animal diseases
viruses
Response element
Human Immunodeficiency Virus Proteins
Down-Regulation
Sequence diversity
HIV Infections
Biology
Response Elements
Microbiology
Article
03 medical and health sciences
Clinical Research
Virology
Genetic variation
Genotype
Genetics
Coding region
Humans
Viral Regulatory and Accessory Proteins
DNA Primers
Genetic diversity
BST2/tetherin
HIV-1 Vpu
virus diseases
Genetic Variation
Nucleic acid amplification technique
biochemical phenomena, metabolism, and nutrition
Phenotype
In vitro
CD4
Rev/RRE
030104 developmental biology
Medical Microbiology
Downregulation
HIV-1
HIV/AIDS
Infection
Nucleic Acid Amplification Techniques
Biotechnology
Subjects
Details
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....7796f4e94bd0c6a460923adef120667b