Consequences of HLA-associated mutations in HIV-1 subtype C Nef on HLA-I downregulation ability.

Identification of CD8+ T lymphocyte (CTL) escape mutations that compromise the pathogenic functions of the Nef protein may be relevant for an HIV-1 attenuation-based vaccine. Previously, HLA-associated mutations 102H, 105R, 108D, and 199Y were individually statistically associated with decreased Nef-mediated HLA-I downregulation ability in a cohort of 298 HIV-1 subtype C infected individuals. In the present study, these mutations were introduced by site-directed mutagenesis into different patient-derived Nef sequence backgrounds of high similarity to the consensus C Nef sequence, and their ability to downregulate HLA-I was measured by flow cytometry in a CEM-derived T cell line. A substantial negative effect of 199Y on HLA-I downregulation and Nef expression was observed, while 102H and 105R displayed negative effects on HLA-I downregulation ability and Nef expression to a lesser extent. The total magnitude of CTL responses in individuals harboring the 199Y mutation was lower than those without the mutation, although this was not statistically significant. Overall, a modest positive relationship between Nef-mediated HLA-I downregulation ability and total magnitude of CTL responses was observed, suggesting that there is a higher requirement for HLA-I downregulation with increased CTL pressure. These results highlight a region of Nef that could be targeted by vaccine-induced CTL to reduce HLA-I downregulation and maximize CTL efficacy.

Modelling and in vitro testing of the HIV-1 Nef fitness landscape.

An effective vaccine is urgently required to curb the HIV-1 epidemic. We have previously described an approach to model the fitness landscape of several HIV-1 proteins, and have validated the results against experimental and clinical data. The fitness landscape may be used to identify mutation patterns harmful to virus viability, and consequently inform the design of immunogens that can target such regions for immunological control. Here we apply such an analysis and complementary experiments to HIV-1 Nef, a multifunctional protein which plays a key role in HIV-1 pathogenesis. We measured Nef-driven replication capacities as well as Nef-mediated CD4 and HLA-I down-modulation capacities of thirty-two different Nef mutants, and tested model predictions against these results. Furthermore, we evaluated the models using 448 patient-derived Nef sequences for which several Nef activities were previously measured. Model predictions correlated significantly with Nef-driven replication and CD4 down-modulation capacities, but not HLA-I down-modulation capacities, of the various Nef mutants. Similarly, in our analysis of patient-derived Nef sequences, CD4 down-modulation capacity correlated the most significantly with model predictions, suggesting that of the tested Nef functions, this is the most important in vivo. Overall, our results highlight how the fitness landscape inferred from patient-derived sequences captures, at least in part, the in vivo functional effects of mutations to Nef. However, the correlation between predictions of the fitness landscape and measured parameters of Nef function is not as accurate as the correlation observed in past studies for other proteins. This may be because of the additional complexity associated with inferring the cost of mutations on the diverse functions of Nef.

Nef-mediated inhibition of NFAT following TCR stimulation differs between HIV-1 subtypes.

Functional characterisation of different HIV-1 subtypes may improve understanding of viral pathogenesis and spread. Here, we evaluated the ability of 345 unique HIV-1 Nef clones representing subtypes A, B, C and D to inhibit NFAT signalling following TCR stimulation. The contribution of this Nef function to disease progression was also assessed in 211 additional Nef clones isolated from unique subtype C infected individuals in early or chronic infection. On average, subtype A and C Nef clones exhibited significantly lower ability to inhibit TCR-mediated NFAT signalling compared to subtype B and D Nef clones. While this observation corroborates accumulating evidence supporting relative attenuation of subtypes A and C that may paradoxically contribute to their increased global prevalence and spread, no significant correlations between Nef-mediated NFAT inhibition activity and clinical markers of HIV-1 infection were observed, indicating that the relationship between Nef function and pathogenesis is complex.