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Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells.

ABSTRACT: G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

SUBMITTER: Frasson I

PROVIDER: S-EPMC8087788 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Herpes simplex virus 1 (HSV-1) transcription is tightly regulated in a temporal cascade, utilizing cellular RNA polymerase. We previously observed that infection with HSV-1 mutants lacking immediate early (IE) genes α0, α4, and α22 exhibited high levels of aberrant transcription across the viral genome at just 1.5 hpi. The strongest effect occurred in the absence of full-length ICP4 which is both an essential transcriptional activator and repressor. The goal of the current study was to define the mechanism of ICP4-mediated early transcriptional repression on the viral genome. Using PRO-Cap, PRO-Seq, GRO-Seq, and Nanopore direct RNA sequencing to analyze viral transcription, we found that initiation was elevated at viral promoters of all temporal classes in the absence of ICP4. Despite higher levels of initiation, transcription of non-IE genes was stalled within gene bodies and did not lead to production of mature mRNA. Thus, ICP4-independent mechanisms limit expression of viral genes that initiate prematurely. GRO-seq data also indicated rapid release from promoter proximal pausing, and rapid progression along HSV IE gene bodies, with termination as an important rate-limiting regulatory step. These findings highlight multiple mechanisms that HSV-1 employs to regulate early transcription and shows that ICP4's repressive role is to restrict initiation, thereby ensuring correct progression of the temporal cascade. IMPORTANCE Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

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Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells.

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