Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation.

The cytoplasmic domain of lentiviral Envelopes (EnvCD) ensures Env incorporation into nascent virions and regulates Env trafficking to and from the plasma membrane. It has also been reported to promote transcription from the viral LTR both directly and indirectly. Noticeably, the HIV-1 and SIVmac239 EnvCDs were described to trigger nuclear translocation of NF-κB (Postler, Cell Host Microbes 2012). Given the paramount importance of identifying viral and host factors regulating HIV transcription, cellular signaling pathways and latency, and given that viral replication capacity is dependent on Env, we asked whether HIV EnvCDs from different HIV-1 subtypes differently modulated NF-κB. To that aim, we evaluated the ability of primary HIV-1 Envs from subtypes B and C to activate the NF-κB pathway. Primary subtype B and C Envs all failed to activate the NF-κB pathway. In contrast, when the EnvCD of HIV-1 Envs was fused to the the CD8-α chain, it induced ~ 10-fold increase in NF-κB induction, and this increase was much stronger with a truncated form of the HIV EnvCD lacking the 76 C-terminal residues and containing the proposed TAK-1 binding domain. Our results indicate that the HIV-1 EnvCD is unlikely to trigger the NF-κB pathway in its native trimeric form.

The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission.

The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines.

Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.