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Variable Nef expression results in differences in Nef-mediated downregulation of MHC I and CD4 between HIV-1 subtypes

The following abstract was presented as part of London Health Research Day 2016

Research Area: Molecular, Cellular, Infection, Immunity, and Cancer Biology
First Author: Aaron Johnson
Supervisor(s): Dr. Jimmy Dikeakos

Introduction:
Since its emergence, HIV-1 has undergone continuous selective pressure leading to a staggering degree of genetic diversity. However, HIV-1 research has largely been limited to only a few subtypes, failing to provide a complete understanding of the epidemic. We aimed to study the consequences of HIV-1 genetic variability on the viral accessory protein Nef. Nef is essential to HIV-1 pathogenesis in part due to its ability to mediate HIV-1 immune evasion and increase viral replication by downregulating cell surface receptors MHC I, and CD4, respectively.

Hypothesis:
I hypothesize that differences in disease progression observed between patients infected with various HIV-1 subtypes will be reflected in differences in Nef function and expression.

Materials and Methods:
In order to investigate Nef function across a diverse range of HIV-1 subtypes we measured cell surface MHC I levels in a human T cell line pseudoinfected with modified HIV-1 viruses expressing Nef proteins from 11 HIV-1 subtypes. CD4 cell surface levels were measured by transfecting CD4+ HeLa cells with expression plasmids encoding Nef-EGFP fusion proteins. MHC I and CD4 downregulation efficiency was measured by flow cytometry. Expression of Nef was analyzed at the mRNA level by qRT-PCR and at the protein level by Western blot. Site-directed mutagenesis and yeast-based recombination were used to generate chimeric Nef proteins.

Results:
Our results demonstrate MHC I and CD4 are differentially downregulated between subtypes. We have identified a subset of low functioning Nef proteins from subtypes C, G and H that are poorly expressed, suggesting a mechanism for decreased receptor downregulation. Decreased Nef expression from these subtypes was consistent between independent expression systems and was not due to differences in transcription, as these subtypes had equivalent mRNA levels to high expressing subtypes. In addition, through generation of chimeric Nef proteins we identified the potential genetic determinants of decreased Nef expression in HIV-1 subtype C, the most prevalent subtype in the HIV/AIDS epidemic.

Discussion and Conclusion:
Our study represents a comprehensive analysis of Nef function among HIV-1 subtypes and suggests differences in expression of the viral accessory protein Nef across HIV-1 subtypes, which may play a role in intersubtype differences in disease progression. Importantly, we have identified a contrast in expression levels of Nef from HIV-1 subtype B and subtype C. Nef from subtype B exhibits high expression and function as opposed to the low expressing and low functioning subtype C. Given the historical bias in HIV/AIDS research towards subtype B and the overwhelming global prevalence of HIV-1 subtype C infections, these findings may influence the treatment of patients as the fight against HIV/AIDS continues.