Natural Killer (NK) cells play an important role in the control of viral infections, recognizing virally infected cells through a variety of activating and inhibitory receptors1–3. Epidemiological and functional studies have recently suggested that NK cells can also contribute to the control of HIV-1 infection through recognition of virally infected cells by both activating and inhibitory Killer Immunoglobulin-like receptors (KIRs)4–7. However, it remains unknown whether NK cells can directly mediate antiviral immune pressure in vivo in humans. Here we describe KIR-associated amino acid polymorphisms in the HIV-1 sequence of chronically infected individuals on a population level. We show that these KIR-associated HIV-1 sequence polymorphisms can enhance the binding of inhibitory KIRs to HIV-1-infected CD4+ T cells, leading to reduced antiviral activity of KIR+ NK cells. These data demonstrate that KIR+ NK cells can place immunological pressure on HIV-1, and that the virus can evade such NK cell mediated immune pressure by selecting for sequence polymorphisms, as previously described for virus-specific T cells and neutralizing antibodies8. NK cells might therefore play a previously underappreciated role in contributing to viral evolution.

Control of human immunodeficiency virus type 1 (HIV-1) by HLA-B27-positive subjects has been linked to an immunodominant CD8؉ cytotoxic T-lymphocyte (CTL) response targeting the conserved KK10 epitope (KRWIILGLNK 263-272 ) in p24/Gag. Viral escape in KK10 typically occurs through development of an R 264 K substitution in conjunction with the upstream compensatory mutation S 173 A, and the difficulty of the virus to escape from the immune response against the KK10 epitope until late in infection has been associated with slower clinical progression. Rare alternative escape mutations at R 264 have been observed, but factors dictating the preferential selection of R 264 K remain unclear. Here we illustrate that while all observed R 264 mutations (K, G, Q, and T) reduced peptide binding to HLA-B27 and impaired viral replication, the replicative defects of the alternative mutants were actually less pronounced than those for R 264 K. Importantly, however, none of these mutants replicated as well as an R 264 K variant containing the compensatory mutation S 173 A. In assessing the combined effects of viral replication and CTL escape using an in vitro coculture assay, we further observed that the compensated R 264 K mutant also displayed the highest replication capacity in the presence of KK10-specific CTLs. Comparisons of codon usage for the respective variants indicated that generation of the R 264 K mutation may also be favored due to a G-to-A bias in nucleotide substitutions during HIV-1 replication. Together, these data suggest that the preference for R 264 K is due primarily to the ability of the S 173 Acompensated virus to replicate better than alternative variants in the presence of CTLs, suggesting that viral fitness is a key contributor for the selection of immune escape variants.

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL 1395-1403 was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.