title: SRPIN340, a cellular serine arginine protein kinases (SRPK) inhibitor, as a potential therapeutic for HPV16 infection
Creators: name: Faizo, Arwa Ali A
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Papillomaviruses, splicing, SRPK, SRPIN340, cervical cancer, therapeutic agent.

description: High-risk human papillomavirus type 16 (HPV16) is the most prevalent HPV infection in the globe. Worldwide, persistent HPV16 infection is responsible for approximately 55-60% of cervical cancer cases, the fourth most common cancer in women with an estimated 570 000 new cases in 2018. The only medical approaches available are prophylactic vaccination and surgical intervention to remove existing lesions. Thus, finding novel antiviral drugs is of a great importance to control HPV16-associated disease progression in the millions infected worldwide. The HPV16 life cycle is tightly linked to epithelial differentiation. During the HPV16 life cycle, cellular splicing plays a major role in the production of viral mRNAs and completion of the replication cycle. Many lines of evidence suggest that HPV16 alters the cellular splicing machinery in order to complete viral replication, particularly in differentiated cells. Splicing involves removal of pre-mRNA introns (non-coding regions) and splicing together of exons (coding regions) to obtain mature mRNA. Serine/Arginine rich (SR) proteins are key regulators of splicing. SR protein kinases (SRPKs) control the activity and localization of SR proteins through phosphorylation of their serine-arginine (RS) domains. Herein, the potential antiviral activity of an SRPK inhibitor, SRPIN340, was evaluated in both HPV16-positive keratinocyte monolayer and 3D organotypic raft cultures. The 3D organotypic raft culture model provided a physiologically relevant system that recapitulated the in vivo micro-environment and supported the natural HPV16 infection biology. Hence, utilization of this model was crucial to accurately evaluate the antiviral efficiency of SRPIN340 during HPV16 infection. SRPIN340 treatment at a concentration of ≤ 50 µM for 48 h did not induce cellular toxicity. Indeed, cellular viability, morphology, growth rate, differentiation and proliferation statuses were unaffected by SRPIN340. However, SRPIN340 treatment led to a significant reduction in the levels of phosphorylated SRSF1, and SRSF3, 4, 6 and 7 while SRSF2 and SRSF5 expression remained unaffected. Furthermore, SRPIN340 localized the nucleo-cytoplasmic shuttling protein SRSF1 and SRSF3 to the cytoplasm of treated cells. On the other hand, SRSF2 nuclear localization remained unaffected. Given the key role of SRSFs in HPV16 infection, the effect of SRPIN340 treatment on viral protein expression was investigated. SRPIN340 treatment significantly inhibited the expression and led to cytoplasmic localization of the viral transcription/replication factor E2. The levels of E2 and E1C transcripts encoding E2 protein were reduced in SRPIN340-treated cells, suggesting that SRPIN340-mediated inhibition of E2 protein expression occurs either at transcriptional or splicing levels. Reduced expression of E2 in SRPIN340-treated cells was also associated with reduced levels of L1 (the major capsid protein) and E4 (a late protein that is likely required for genome amplification and virion release). Importantly, however, the inhibitory effect of SRPIN340 on these viral proteins did not induce cellular tumorigenesis, as demonstrated by normal levels of viral oncoproteins E6 and E7 and cellular tumour suppressor markers p53 and Rb. The underlying mechanism for SRPIN340 antiviral activity is not entirely clear. Yet, genetic knock-down of SRSF3 and to a lesser extent SRSF5, but not other SRSFs, led to a significant reduction of E2 expression. Given that SRPIN340 did not affect phosph-SRSF5 expression, this study provides an insight on how SRPIN340 might affect viral protein expression through regulation of SRSF3 expression and subcellular localization. Collectively, these data demonstrate for the first time the antiviral potency of SRPIN340 treatment during HPV16 infection in both 2D and 3D culture models at optimized treatment conditions that do not induce cellular toxicity. Moreover, they highlight the significance of SR proteins as therapeutic targets during viral infections that are known to hijack the cellular splicing machinery in order to promote their replication.