Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

Jaclyn K. Mann; Helen Byakwaga; Xiaomei T. Kuang; Anh Q. Le; Chanson J. Brumme; Philip Mwimanzi; Saleha Omarjee; Eric Martin; Guinevere Q. Lee; Bemuluyigza Baraki; Ryan Danroth; Rosemary McCloskey; Conrad Muzoora; David R. Bangsberg; Peter W. Hunt; Philip J.R. Goulder; Bruce D. Walker; P. R. Harrigan; Jeff N. Martin; Thumbi Ndung'u; Mark A. Brockman; Zabrina L. Brumme

doi:10.1186/1742-4690-10-100

Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

Jaclyn K. Mann, Helen Byakwaga, Xiaomei T. Kuang, Anh Q. Le, Chanson J. Brumme, Philip Mwimanzi, Saleha Omarjee, Eric Martin, Guinevere Q. Lee, Bemuluyigza Baraki, Ryan Danroth, Rosemary McCloskey, Conrad Muzoora, David R. Bangsberg, Peter W. Hunt, Philip J.R. Goulder, Bruce D. Walker, P. R. Harrigan, Jeff N. Martin, Thumbi Ndung'uMark A. Brockman, Zabrina L. Brumme

Research output: Contribution to journal › Article › peer-review

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Abstract

Background: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.Results: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.Conclusions: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

Original language	English (US)
Article number	100
Journal	Retrovirology
Volume	10
Issue number	1
DOIs	https://doi.org/10.1186/1742-4690-10-100
State	Published - Sep 16 2013
Externally published	Yes

Keywords

CD4
HIV/AIDS
HLA class I
Nef
Pathogenesis
Viral diversity

ASJC Scopus subject areas

Virology
Infectious Diseases

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10.1186/1742-4690-10-100

Cite this

Mann, J. K., Byakwaga, H., Kuang, X. T., Le, A. Q., Brumme, C. J., Mwimanzi, P., Omarjee, S., Martin, E., Lee, G. Q., Baraki, B., Danroth, R., McCloskey, R., Muzoora, C., Bangsberg, D. R., Hunt, P. W., Goulder, P. J. R., Walker, B. D., Harrigan, P. R., Martin, J. N., ... Brumme, Z. L. (2013). Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes. Retrovirology, 10(1), Article 100. https://doi.org/10.1186/1742-4690-10-100

Mann, JK, Byakwaga, H, Kuang, XT, Le, AQ, Brumme, CJ, Mwimanzi, P, Omarjee, S, Martin, E, Lee, GQ, Baraki, B, Danroth, R, McCloskey, R, Muzoora, C, Bangsberg, DR, Hunt, PW, Goulder, PJR, Walker, BD, Harrigan, PR, Martin, JN, Ndung'u, T, Brockman, MA & Brumme, ZL 2013, 'Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes', Retrovirology, vol. 10, no. 1, 100. https://doi.org/10.1186/1742-4690-10-100

@article{6a626ce7714141cf8c45024d0627e7a5,

title = "Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes",

abstract = "Background: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.Results: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-na{\"i}ve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.Conclusions: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.",

keywords = "CD4, HIV/AIDS, HLA class I, Nef, Pathogenesis, Viral diversity",

author = "Mann, {Jaclyn K.} and Helen Byakwaga and Kuang, {Xiaomei T.} and Le, {Anh Q.} and Brumme, {Chanson J.} and Philip Mwimanzi and Saleha Omarjee and Eric Martin and Lee, {Guinevere Q.} and Bemuluyigza Baraki and Ryan Danroth and Rosemary McCloskey and Conrad Muzoora and Bangsberg, {David R.} and Hunt, {Peter W.} and Goulder, {Philip J.R.} and Walker, {Bruce D.} and Harrigan, {P. R.} and Martin, {Jeff N.} and Thumbi Ndung'u and Brockman, {Mark A.} and Brumme, {Zabrina L.}",

note = "Funding Information: We thank Dr. Art FY Poon for assistance with HIV-1 sequence alignments. This work was supported by operating grants from the Canadian Institutes for Health Research (CIHR) (MOP-93536 and HOP-115700, to ZLB and MAB). JKM and HB received a pilot grant from the Canada-Sub Saharan Africa (CANSSA) HIV/AIDS Network through funding provided by the Global Health Research Initiative (GHRI), itself a collaborative research funding partnership of the CIHR, the Canadian International Development Agency (CIDA), and the International Development Research Centre (IDRC). CJB is supported by a Vanier Canada Graduate Scholarship from CIHR. EM was supported by a Master{\textquoteright}s Scholarship from the Canadian Association for HIV Research and Abbott Virology. RD is supported by the Merck-Canada Training of Aboriginal Youth in Biomedical Labs program, while RMM received a Vice President Research-Undergraduate Student Research Award, both at Simon Fraser University. TN holds the South African DST/NRF Research Chair in Systems Biology of HIV/AIDS, the Victor Daitz Chair in HIV/TB Research and an International Early Career Scientist Award from the Howard Hughes Medical Institute. MAB holds a Canada Research Chair in Viral Pathogenesis and Immunity. ZLB is the recipient of a CIHR New Investigator Award and a Scholar Award from the Michael Smith Foundation for Health Research. The funders played no role in the study design; collection, analysis, or interpretation of data; or writing of this manuscript.",

year = "2013",

month = sep,

day = "16",

doi = "10.1186/1742-4690-10-100",

language = "English (US)",

volume = "10",

journal = "Retrovirology",

issn = "1742-4690",

publisher = "BioMed Central",

number = "1",

}

TY - JOUR

T1 - Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

AU - Mann, Jaclyn K.

AU - Byakwaga, Helen

AU - Kuang, Xiaomei T.

AU - Le, Anh Q.

AU - Brumme, Chanson J.

AU - Mwimanzi, Philip

AU - Omarjee, Saleha

AU - Martin, Eric

AU - Lee, Guinevere Q.

AU - Baraki, Bemuluyigza

AU - Danroth, Ryan

AU - McCloskey, Rosemary

AU - Muzoora, Conrad

AU - Bangsberg, David R.

AU - Hunt, Peter W.

AU - Goulder, Philip J.R.

AU - Walker, Bruce D.

AU - Harrigan, P. R.

AU - Martin, Jeff N.

AU - Ndung'u, Thumbi

AU - Brockman, Mark A.

AU - Brumme, Zabrina L.

N1 - Funding Information: We thank Dr. Art FY Poon for assistance with HIV-1 sequence alignments. This work was supported by operating grants from the Canadian Institutes for Health Research (CIHR) (MOP-93536 and HOP-115700, to ZLB and MAB). JKM and HB received a pilot grant from the Canada-Sub Saharan Africa (CANSSA) HIV/AIDS Network through funding provided by the Global Health Research Initiative (GHRI), itself a collaborative research funding partnership of the CIHR, the Canadian International Development Agency (CIDA), and the International Development Research Centre (IDRC). CJB is supported by a Vanier Canada Graduate Scholarship from CIHR. EM was supported by a Master’s Scholarship from the Canadian Association for HIV Research and Abbott Virology. RD is supported by the Merck-Canada Training of Aboriginal Youth in Biomedical Labs program, while RMM received a Vice President Research-Undergraduate Student Research Award, both at Simon Fraser University. TN holds the South African DST/NRF Research Chair in Systems Biology of HIV/AIDS, the Victor Daitz Chair in HIV/TB Research and an International Early Career Scientist Award from the Howard Hughes Medical Institute. MAB holds a Canada Research Chair in Viral Pathogenesis and Immunity. ZLB is the recipient of a CIHR New Investigator Award and a Scholar Award from the Michael Smith Foundation for Health Research. The funders played no role in the study design; collection, analysis, or interpretation of data; or writing of this manuscript.

PY - 2013/9/16

Y1 - 2013/9/16

N2 - Background: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.Results: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.Conclusions: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

AB - Background: The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.Results: Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.Conclusions: Results support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

KW - CD4

KW - HIV/AIDS

KW - HLA class I

KW - Nef

KW - Pathogenesis

KW - Viral diversity

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U2 - 10.1186/1742-4690-10-100

DO - 10.1186/1742-4690-10-100

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AN - SCOPUS:84883810916

SN - 1742-4690

VL - 10

JO - Retrovirology

JF - Retrovirology

IS - 1

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ER -

Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

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