Masters Degree. University of KwaZulu-Natal, Durban.HIV-1 Nef is a small accessory protein that plays a vital role in enhancing HIV-1 pathogenesis,
evidenced by a strongly attenuated disease course following infection with a virus with gross
Nef defects. Nef has multiple cellular effects, which enhance HIV-1 replication and immune
evasion. Major activities of Nef include CD4 down-regulation, HLA-I down-regulation, and
CD4-independent enhancement of virion infectivity. Recent studies have uncovered Nefmediated
down-regulation of the host restriction factor SERINC5 as an important mechanism
by which Nef enhances virion infectivity. However, there is a lack of studies defining the role
of this function in HIV-1 pathogenesis. Previous studies indicated that Nef-mediated CD4
down-regulation and enhancement of infectivity are likely the major contributors to Nef’s
effect of enhancing pathogenicity; the relative significance of each Nef function for HIV-1
disease progression remains incompletely understood.
Given the key role of Nef-mediated SERINC5 down-regulation in enhancing virion infectivity,
the primary aim of the present study was to determine if this Nef activity contributes
significantly to disease progression in individuals infected with HIV-1 subtype C, which is the
dominant HIV-1 subtype worldwide. To investigate this, SERINC5 down-regulation activity
of 106 Nef clones derived from patients with early HIV-1 subtype C infection were evaluated
in a CD4+ T cell line using a flow cytometry-based assay and subsequently related to viral load
set point and to the rate of CD4+ T cell decline using linear regression analysis. The second
aim of this study was to assess the overall contribution of SERINC5 down-regulation to Nef
function, using linear regression analysis with E values as a proxy for overall Nef function in
vivo.
The third aim of the study was to identify amino acid variants that significantly alter Nefmediated
SERINC5 down-regulation using a codon-by-codon sequence-function analysis tool
available online.
No significant relationship was found between each Nef function and viral set point (SERINC5
down-regulation, p=0.28) or rate of CD4+ T cell decline (SERINC5 down-regulation, p=0.48).
CD4 down-regulation (p=0.02) and SERINC5 down-regulation (p=0.003) were significant
determinants of the E value in univariate analyses, and SERINC5 down-regulation remained
significant in the multivariate analysis (p=0.003). We found several amino acids that were
significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or
decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity.
In conclusion, none of the Nef functions in our study, including SERINC5 down-regulation,
were found to be significant individual contributors to disease progression. However,
interestingly we found CD4 down-regulation and SERINC5 down-regulation to be the largest
contributors, of the Nef functions considered here, to overall Nef function and that the
contribution of SERINC5 down-regulation was the most significant. Taken together, this could
be explained by multiple Nef functions acting together to facilitate the enhancement of viral
spread and immune evasion in vivo that ultimately enhance disease progression. We found
several amino acid variants that either increased or decreased Nef’s ability to down-regulate
SERINC5; however, further studies in the form of site-directed mutagenesis are warranted to
further understand their effect on SERINC5 down-regulation activity. In summary, the results
suggest that SERINC5 down-regulation is a strong contributor to overall Nef function and
identifies potential genetic determinants of this Nef function that may have relevance for
vaccines or therapeutics
