Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.
HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24(Gag) (GQMVHQAIGag140-147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13.


OBJECTIVE
Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.
Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada.
Aniqa
A
Shahid
IrsiCaixa AIDS Research Institute, HIVACAT, UAB, Barcelona, Spain.
Alex
A
Olvera
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada.
Gursev
G
Anmole
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada.
Xiaomei T
XT
Kuang
Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada.
Laura A
LA
Cotton
AIDS Research Group, IDIBAPS, HIVACAT, Hospital Clinic, UAB, Barcelona, Spain.
Montserrat
M
Plana
IrsiCaixa AIDS Research Institute, HIVACAT, UAB, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain University of Vic and Central University of Catalonia, Vic, Barcelona, Spain.
Christian
C
Brander
Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
Mark A
MA
Brockman
Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada zbrumme@sfu.ca.
Zabrina L
ZL
Brumme
D002460:Cell Line; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D016655:HIV Core Protein p24 / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D059908:HLA-B13 Antigen / Q000235:genetics*; D006801:Humans; D057131:Immune Evasion / Q000235:genetics* / Q000276:immunology; D011110:Polymorphism, Genetic / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics*; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D018984:Epitopes, T-Lymphocyte; D016655:HIV Core Protein p24; D059908:HLA-B13 Antigen; D054301:gag Gene Products, Human Immunodeficiency Virus; D054311:nef Gene Products, Human Immunodeficiency Virus; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D054302:pol Gene Products, Human Immunodeficiency Virus