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-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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada. mark_brockman@sfu.ca.
authors.forename: Jaclyn K
authors.initials: JK
authors.lastname: Mann
authors.forename: Helen
authors.initials: H
authors.lastname: Byakwaga
authors.forename: Xiaomei T
authors.initials: XT
authors.lastname: Kuang
authors.forename: Anh Q
authors.initials: AQ
authors.lastname: Le
authors.forename: Chanson J
authors.initials: CJ
authors.lastname: Brumme
authors.forename: Philip
authors.initials: P
authors.lastname: Mwimanzi
authors.forename: Saleha
authors.initials: S
authors.lastname: Omarjee
authors.forename: Eric
authors.initials: E
authors.lastname: Martin
authors.forename: Guinevere Q
authors.initials: GQ
authors.lastname: Lee
authors.forename: Bemuluyigza
authors.initials: B
authors.lastname: Baraki
authors.forename: Ryan
authors.initials: R
authors.lastname: Danroth
authors.forename: Rosemary
authors.initials: R
authors.lastname: McCloskey
authors.forename: Conrad
authors.initials: C
authors.lastname: Muzoora
authors.forename: David R
authors.initials: DR
authors.lastname: Bangsberg
authors.forename: Peter W
authors.initials: PW
authors.lastname: Hunt
authors.forename: Philip J R
authors.initials: PJ
authors.lastname: Goulder
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Walker
authors.forename: P Richard
authors.initials: PR
authors.lastname: Harrigan
authors.forename: Jeff N
authors.initials: JN
authors.lastname: Martin
authors.forename: Thumbi
authors.initials: T
authors.lastname: Ndung'u
authors.forename: Mark A
authors.initials: MA
authors.lastname: Brockman
authors.forename: Zabrina L
authors.initials: ZL
authors.lastname: Brumme
mesh_terms: D000328:Adult; D000349:Africa; D015704:CD4 Antigens / Q000096:biosynthesis*; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D015536:Down-Regulation; D005260:Female; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis*; D054884:Host-Pathogen Interactions; D006801:Humans; D008297:Male; D009656:North America; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D015395:Histocompatibility Antigens Class I; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1
references.citation: J Infect Dis. 2007 Apr 15;195(8):1177-80
references.pmid: 17357054
references.citation: EMBO Rep. 2001 Jul;2(7):580-5
references.pmid: 11463741
references.citation: Viruses. 2012 Sep;4(9):1711-30
references.pmid: 23170180
references.citation: J Infect Dis. 1999 Jan;179(1):68-73
references.pmid: 9841824
references.citation: Mol Biol Evol. 2010 Apr;27(4):819-32
references.pmid: 19955476
references.citation: AIDS. 2011 Jul 31;25(12):1515-22
references.pmid: 21505307
references.citation: J Biol Chem. 2003 Sep 5;278(36):33912-9
references.pmid: 12816953
references.citation: AIDS Res Hum Retroviruses. 2012 Dec;28(12):1617-26
references.pmid: 22583022
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11229-34
references.pmid: 9736718
references.citation: J Virol. 2003 Jan;77(2):1589-94
references.pmid: 12502873
references.citation: PLoS Pathog. 2008 Aug 22;4(8):e1000131
references.pmid: 18725938
references.citation: Retrovirology. 2011 Jun 22;8:50
references.pmid: 21696586
references.citation: J Antimicrob Chemother. 2003 Feb;51(2):229-40
references.pmid: 12562686
references.citation: PLoS Pathog. 2007 Jul;3(7):e94
references.pmid: 17616974
references.citation: Virology. 2013 May 10;439(2):74-80
references.pmid: 23490051
references.citation: J Acquir Immune Defic Syndr. 2004 Aug 15;36(5):1100-2
references.pmid: 15247564
references.citation: J Virol. 2012 Jul;86(13):7126-35
references.pmid: 22553319
references.citation: Retrovirology. 2012 May 31;9:47
references.pmid: 22651890
references.citation: Curr Biol. 1999 Jun 17;9(12):613-21
references.pmid: 10375525
references.citation: J Virol. 2008 Sep;82(17):8422-30
references.pmid: 18562530
references.citation: PLoS One. 2009 Aug 19;4(8):e6687
references.pmid: 19690614
references.citation: PLoS One. 2013 Aug 28;8(8):e71758
references.pmid: 24015191
references.citation: Science. 1995 Nov 10;270(5238):988-91
references.pmid: 7481804
references.citation: Retrovirology. 2008 Sep 22;5:84
references.pmid: 18808677
references.citation: AIDS. 1999 May 28;13(8):901-7
references.pmid: 10371170
references.citation: Immunity. 1999 Jun;10(6):661-71
references.pmid: 10403641
references.citation: N Engl J Med. 1995 Jan 26;332(4):228-32
references.pmid: 7808489
references.citation: Biochem Biophys Res Commun. 2001 Jun 15;284(3):638-42
references.pmid: 11396948
references.citation: Retrovirology. 2013 Jan 07;10:1
references.pmid: 23289738
references.citation: Retrovirology. 2012 Apr 27;9:34
references.pmid: 22537596
references.citation: J Virol. 2000 Apr;74(8):3740-51
references.pmid: 10729149
references.citation: J Neuroimmune Pharmacol. 2011 Jun;6(2):230-46
references.pmid: 21336563
references.citation: J Acquir Immune Defic Syndr. 2010 Jun;54(2):180-4
references.pmid: 20010433
references.citation: J Virol. 2003 Jan;77(2):1021-38
references.pmid: 12502818
references.citation: J Virol. 2010 Oct;84(20):10820-31
references.pmid: 20702636
references.citation: J Virol. 2000 Nov;74(21):9836-44
references.pmid: 11024110
references.citation: Syst Biol. 2003 Oct;52(5):696-704
references.pmid: 14530136
references.citation: Retrovirology. 2010 Oct 12;7:85
references.pmid: 20939905
references.citation: J Int AIDS Soc. 2010 Feb 03;13:4
references.pmid: 20205896
references.citation: Proc Natl Acad Sci U S A. 2001 May 22;98(11):6062-7
references.pmid: 11353856
references.citation: Immunity. 1998 Apr;8(4):483-95
references.pmid: 9586638
references.citation: J Virol. 2004 Dec;78(23):13335-44
references.pmid: 15542684
references.citation: Proc Natl Acad Sci U S A. 2012 Aug 14;109(33):13353-8
references.pmid: 22826228
references.citation: AIDS. 2011 Nov 13;25(17):2123-31
references.pmid: 21881481
references.citation: J Virol. 2001 Apr;75(8):3657-65
references.pmid: 11264355
references.citation: Cell. 1991 May 17;65(4):651-62
references.pmid: 2032289
references.citation: J Biol Chem. 2010 Nov 12;285(46):35350-8
references.pmid: 20833716
references.citation: N Engl J Med. 2008 Apr 10;358(15):1590-602
references.pmid: 18403767
references.citation: J Virol. 2002 May;76(9):4625-33
references.pmid: 11932428
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9440-5
references.pmid: 12883005
references.citation: Lancet Infect Dis. 2011 Jan;11(1):45-56
references.pmid: 21126914
references.citation: J Virol. 1995 Jan;69(1):93-100
references.pmid: 7983771
references.citation: Biochemistry. 1996 Aug 13;35(32):10256-61
references.pmid: 8756680
references.citation: J Virol. 2009 Jun;83(11):5592-605
references.pmid: 19297481
references.citation: AIDS Res Hum Retroviruses. 2009 Mar;25(3):285-96
references.pmid: 19327048
references.citation: Bioinformatics. 2005 Mar 1;21(5):676-9
references.pmid: 15509596
references.citation: AIDS. 2004 Aug 20;18(12):1629-36
references.pmid: 15280773
references.citation: Nat Struct Mol Biol. 2012 Jun 17;19(7):701-6
references.pmid: 22705789
references.citation: AIDS Res Hum Retroviruses. 1999 Jan 1;15(1):23-32
references.pmid: 10024049
references.citation: J Virol. 1999 Mar;73(3):1964-73
references.pmid: 9971776
references.citation: J Virol. 2000 Mar;74(6):2907-12
references.pmid: 10684310
references.citation: J Virol. 1999 Jul;73(7):5497-508
references.pmid: 10364298