The RNA-Binding Proteins SRP14 and HMGB3 Control HIV-1 Tat mRNA Processing and Translation During HIV-1 Latency

Abstract

HIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4+ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4+ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets.

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency

“…More recently, it was shown that several RNA binding proteins can regulate Tat RNA processing and translation by binding to an RNA regulatory element (referred to as Tat IRES modulator of tat mRNA (TIM TAM)) present in Tat-containing transcripts. This study showed that host RNA binding proteins SRP14 (splicing regulator) and HMGB3 bind TIM TAM and modulate Tat gene expression and function and can act as positive (SRP14) or negative (HMGB3) regulators of HIV-1 viral production ( Figure 2A ) ( Khoury et al., 2021 ). Recent transcriptomics and proteomics studies have also identified several putative splicing regulators in reactivation of HIV-1 latency (see section 2).…”

“…In summary, mechanisms that control HIV-1 latency and latency reactivation include regulation of HIV-1 vRNA splicing by modulating the binding of host spliceosome complex factors to nascent HIV-1 vRNA or by acting on HIV-1 vRNA regulatory elements ( Erkelenz et al., 2015 ; Khoury et al., 2018 ; Kyei et al., 2018 ; Yukl et al., 2018 ; Norton et al., 2019 ; Telwatte et al., 2019 ; Khoury et al., 2021 ).…”

“…This block in export of MS HIV-1 vRNA species can be reversed by modulating the presence or absence of nucleocytoplasmic export regulators. For instance, overexpression of protein Polypyrimidine tract-binding protein (PTB)1 ( Figure 2B ) leads to increased export of tat-rev containing mRNAs ( Lassen et al., 2006 ), although its exact mechanism of action and contribution to HIV-1 latency remains unclear ( Khoury et al., 2021 ). Still, once the total amount of HIV-1 Tat and Rev increases over a certain threshold, the restored Tat-rev positive feedback loop facilitates transcription, export, and production of viral particles.…”

Expansion Segment ES30L enriched in birds and mammals can potentially regulate protein synthesis

“…For instance, PCBP1/2 along with hnRNPK have been shown (87, 89) to stimulate IRES-mediated translation of c-myc mRNA in vitro in rabbit reticulocyte lysates. Other proteins from our analysis like SRSF1 (90), SRSF6 (94), YTHDC2 (95), NCL (96, 97), IGF2BP1 (98), SRP14(99), RBM4 (100, 101) have also been shown to activate translation of their cognate mRNAs. Conversely, other proteins from our data like DDX21 (102) and hnRNPK (103) have been shown to inhibit viral IRESs and negatively regulate translation in vivo in mammalian cell lines.…”

The DNA binding high mobility group box protein family functionally binds RNA

“…HMGB1 binds RNA in vivo with an identified motif of 5 0 -MWGRA-3 0 (M = A/C, W = A/U, R = A/G; Sofiadis et al, 2021). From the same subfamily as HMGB1, HMGB3 binds to specific sites within HIV-1 Tat mRNA, including stem loops and internal bulges (Khoury et al, 2021). Many nonhuman HMGBs also bind RNA in vitro (Table 2).…”