Consequences of HLA-B*13-associated escape mutations on HIV-1 replication and Nef protein function
Background: HLA-B*13, a
protective HLA class I allele, selects CTL escape mutations across HIV-1, but
their effects on viral replication and protein function remain incompletely understood.
We assessed the impact of 10 published HLA-B*13 escape mutations in Gag, Pol
and Nef on viral replication. We also assessed the impact of Nef mutations on
cell-surface CD4 and HLA class I downregulation, and the latter''s consequence
for recognition of virus-infected cells by epitope-specific T-cells.
Methods: HLA-B*13 escape
mutations in Gag (A146S, I147L, K436R, I437L), Pol (Protease-L63S; RT-Q334N,
T369A, K374R) and Nef (E24Q, Q107R) were engineered alone and in biologically
relevant combinations (defined via analysis of longitudinal HIV-1 RNA sequences
from 9 B*13+ seroconverters and cross-sectional sequences from 69 B*13+ chronic
patients) into an HIV-1NL4-3 backbone. Viral replication was determined
using a GFP-reporter T-cell assay. Nef-mediated CD4 and HLA-A*02 downregulation
was assessed by flow cytometry. Recognition of infected target cells by HIV-1-specific
effector cells was assessed via co-culture with an NFAT-driven luciferase
reporter T-cell line specific for the A*02-restricted Gag-FK10 peptide.
Results: Of
all mutations tested, only Gag I437L incurred a 14% replicative reduction, alone
and in combination with A146S and/or I147L. This defect was rescued to
wild-type (HIV-1NL4-3) levels by K436R. A novel B*13 epitope was identified in p24Gag
(GQMVHQAI140-147). Single Nef mutations did
not affect CD4
or HLA-A*02 downregulation; however, the Nef double mutant was nearly 70% impaired
for the latter function (for context, the canonical Nef M20ANL4-3 mutant
defective for HLA downregulation was >90% functionally impaired). Correspondingly,
luciferase signal emitted by HIV-1-specific effector cells upon co-culture with
HIV-1E24Q/Q107R-infected target cells was 2-fold higher than for
cells infected with NL4-3 or single Nef mutants.
Conclusions: A
minority of HLA-B*13-driven escape mutations modestly dampen HIV-1 replication or
Nef function, which could contribute in part to B*13-associated protection from
disease progression. The observation that a naturally-occurring (albeit rare;
4%) Nef double mutation impairs HLA downregulation and enhances recognition of
infected cells by HIV-1-specific T-cells suggests a novel escape-associated
defect that ironically dampens a key viral immune evasion strategy. Improved understanding
of the mechanisms underlying HLA-associated protective effects may have
implications for HIV-1 vaccine design.
A. Shahid1, X.T. Kuang2, G. Anmole2, A. Olvera3, L. Cotton1, C. Brander3,4, M.A. Brockman1,2,5, Z.L. Brumme1,5
1Simon Fraser University, Faculty of Health Sciences, Burnaby, Canada, 2Simon Fraser University, Department of Molecular Biology and Biochemistry, Burnaby, Canada, 3Irsicaixa AIDS Research Institute, Barcelona, Spain, 4ICREA, Barcelona, Spain, 5BC Centre for Excellence in HIV/AIDS, Vancouver, Canada