Control of human papillomavirus gene expression by alternative splicing

Human papillomaviruses possess circular double stranded DNA genomes of around 8 kb in size from which multiple mRNAs are synthesized during an infectious life cycle. Although at least three viral promoters are used to initiate transcription, viral mRNAs are largely the product of processing of pre-mRNAs by alternative splicing and polyadenylation. The HPV life cycle and viral gene expression are tightly linked to differentiation of the epithelium the virus infects: there is an orchestrated production of viral mRNAs and proteins. In this review we describe viral mRNA expression and the roles of the SR and hnRNP proteins that respectively positively and negatively regulate splicing. We discuss HPV regulation of splicing factors and detail the evidence that the papillomavirus E2 protein has splicing-related activities. We highlight the possibility that HPV-mediated control of splicing in differentiating epithelial cells may be necessary to accomplish the viral replication cycle

Human papillomavirus type 16 infection activates the host serine arginine protein kinase 1 (SRPK1) - splicing factor axis

The infectious life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Evidence suggests a sophisticated interplay between host gene regulation and virus replication. Alternative splicing is an essential process for host and viral gene expression, and is generally upregulated by serine arginine-rich splicing factors (SRSFs). SRSF activity can be positively or negatively controlled by cycles of phosphorylation/dephosphorylation. Here we show that HPV16 infection leads to accumulation of the paradigm SRSF protein, SRSF1, in the cytoplasm in a keratinocyte differentiation-specific manner. Moreover, HPV16 infection leads to increased levels of cytoplasmic and nuclear phosphorylated SRSF1. SR protein kinase 1 (SRPK1) phosphorylates SRSF1. Similar to HPV upregulation of SRSF1, we demonstrate HPV upregulation of SRPK1 via the viral E2 protein. SRPK1 depletion or drug inhibition of SRPK1 kinase activity resulted in reduced levels of SRSF1, suggesting that phosphorylation stabilizes the protein in differentiated HPV-infected keratinocytes. Together, these data indicate HPV infection stimulates the SRPK1–SRSF axis in keratinocytes

High Genetic Diversity of Human Rhinovirus among Pilgrims with Acute Respiratory Tract Infections during 2019 Hajj Pilgrimage Season

OBJECTIVES: Acute Respiratory tract infections (ARI) due to Human Rhinoviruses (HRV) are common in pilgrims during the annual Hajj pilgrimage. The objective of this study was to investigate the genetic diversity of HRV among pilgrims with respiratory symptoms during Hajj 2019. METHODS: HRV infection was detected using multiplex real time RT-PCR. Cycle sequencing was performed on positive samples and the sequences were subjected to phylogenetic analysis. RESULTS: 19 HRV-positive respiratory samples were sequenced. All three serotypes of HRV were identified: HRV-A (13; [68.42%)) was more common than HRV-B (2; [10.53%]), and HRV-C (4; [21.05%]). HRV-A species were found to be of genotypes A101, A21, A30, A57, A23, A60 and A11. HRV-B species belonged to genotypes B4 and B84, and HRV-C species were of genotypes C15, C3 and C56. CONCLUSIONS: Sequencing studies of respiratory tract viruses in pilgrims are important. We provide preliminary evidence of high diversity of HRV genotypes circulating in pilgrims in a restricted area during Hajj. This requires further clinical and sequencing studies of viral pathogens in larger consorts of overseas and local pilgrims

SRPIN340, a cellular serine arginine protein kinases (SRPK) inhibitor, as a potential therapeutic for HPV16 infection

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

Development of Serological Assays and Seroprevalence Studies of the New Coronavirus 2019 (COVID-19): Reports from Saudi Arabia

Serological assays are valuable tools for tracking COVID-19 spread, estimation of herd immunity, and evaluation of vaccine effectiveness. Several reports from Saudi Arabia describe optimized in-house protocols that enable detection of SARS-CoV-2 specific antibodies and measurement of their neutralizing activity. Notably, there were variations in the approaches utilized to develop and validate these immunoassays in term of sample size, validation methodologies, and statistical analyses. The developed enzyme-linked immunoassays (ELISAs) were based on the viral full-length spike (S), S1 subunit, and nucleocapsid (NP), and enabled detection of IgM and/or IgG. ELISAs were evaluated and validated against a microneutralization assay utilizing a local SARS-CoV-2 clinical isolate, FDA-approved commercially available immunoassays, and/or real-time polymerase chain reaction (RT-PCR). Overall, the performance of the described assays was high, reaching up to 100% sensitivity and 98.9% specificity with no cross-reactivity with other coronaviruses. In-house immunoassays, along with commercially available kits, were subsequently applied in a number of sero-epidemiological studies aiming to estimate sero-positivity status among local populations including healthcare workers, COVID-19 patients, non-COVID-19 patients, and healthy blood donors. The reported seroprevalence rates differed widely among these studies, ranging from 0.00% to 32.2%. These variations are probably due to study period, targeted population, sample size, and performance of the immunoassays utilized. Indeed, lack of sero-positive cases were reported among healthy blood donors during the lockdown, while the highest rates were reported when the number of COVID-19 cases peaked in the country, particularly among healthcare workers working in referral hospitals and quarantine sites. In this review, we aim to (1) provide a critical discussion about the developed in-house immunoassays, and (2) summarize key findings of the sero-epidemiological studies and highlight strengths and weaknesses of each study

Coronavirus disease of 2019 (COVID-19) in the Gulf Cooperation Council (GCC) countries: Current status and management practices

Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global threat to human population. The numbers of cases and deaths due to COVID-19 are escalating daily, putting health care systems worldwide under tremendous pressure. Policymakers in the affected countries have adopted varying strategies to deal with this crisis. As a result, the current COVID-19 status in terms of number of cases and deaths hugely varies between countries. The Gulf Cooperation Council (GCC) countries have managed to limit the massive spread of the infection among their populations by implementing proactive plans and timely decisions in response to COVID-19 outbreak; measures taken included suspension of flights, closure of educational institutes, curfew and lockdown of major cities, and provision of free-of-charge healthcare to patients. This review summarizes the COVID-19 status as of 18 May 2020 and highlights prevention and control measures applied in the GCC countries

Native ligand (X77)-protein (PDB structure 6W63) 2D interactions plot: Representation of intermolecular interaction between native ligand and the active site amino acid residue of the target protein.

Native ligand (X77)-protein (PDB structure 6W63) 2D interactions plot: Representation of intermolecular interaction between native ligand and the active site amino acid residue of the target protein.</p

Fig 7 -

Root mean square deviation (RMSD) plots for (a) Mpro-CHEMBL1940602 (b) Mpro-CHEMBL2036486 (c) Mpro-CHEMBL3628485 (d) Mpro-CHEMBL200972 (e) Mpro-CHEMBL2036488, and (fCH) Mpro-X77 (control), calculated over the period of 100 ns MD simulation.</p

Binding scores of compounds were obtained via three different accuracy levels applied during the structure-based virtual screening process.

(a) HTVS, (2) Glide SP, and (c) Glide XP.</p

S2 File -

(DOCX)</p