TY - JOUR
T1 - In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
AU - Rahimi, Asa
AU - Anmole, Gursev
AU - Soto-Nava, Maribel
AU - Escamilla-Gomez, Tania
AU - Markle, Tristan
AU - Jin, Steven W.
AU - Lee, Guinevere Q.
AU - Harrigan, P. Richard
AU - Bangsberg, David R.
AU - Martin, Jeffrey
AU - Avila-Rios, Santiago
AU - Reyes-Teran, Gustavo
AU - Brockman, Mark A.
AU - Brumme, Zabrina L.
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2017/2/1
Y1 - 2017/2/1
N2 - 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.
AB - 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.
KW - BST2/tetherin
KW - CD4
KW - Downregulation
KW - HIV-1 Vpu
KW - Rev/RRE
KW - Sequence diversity
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UR - http://www.scopus.com/inward/citedby.url?scp=84997221464&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2016.11.004
DO - 10.1016/j.jviromet.2016.11.004
M3 - Article
C2 - 27865749
AN - SCOPUS:84997221464
SN - 0166-0934
VL - 240
SP - 32
EP - 41
JO - Journal of Virological Methods
JF - Journal of Virological Methods
ER -