Sheila V. Graham

Hyperthermia is a well-documented adjunct to cancer therapy. Recently, it has begun to gain traction in the treatment of human papillomavirus (HPV)-driven pathologies. Microwaves offer a precise and targeted approach to deliver hyperthermia and are already in use in the clinic for the treatment of plantar and common warts: non-oncogenic HPV-driven cutaneous lesions. To determine if microwave-delivered hyperthermia may be applicable for the treatment of oncogenic HPV-positive anogenital mucosal lesions and to better understand the molecular mechanisms underlying microwave-delivered hyperthermia. Three-dimensional in vitro grown HPV16-infected epithelial tissues were subject to hyperthermia using microwave treatment. RNA sequencing was used to examine changes to the transcriptome following microwave treatment. Microwave treatment rapidly altered the transcriptome, with 59 genes differentially expressed at 4 h post-treatment. Of these genes, three were downregulated and 56 upregulated. At later time points post-treatment, 150 genes were differentially expressed, and all of these were upregulated. Microwave treatment triggered a heat shock response, with many chaperones and co-chaperones upregulated, which are important in preventing proteolysis following temperature rises. Significant upregulation of many of these genes has previously been reported in transcriptomic studies that used other methods of delivering hyperthermia. Microwave hyperthermia increased expression of genes involved in immune pathways, including interleukins (IL8, IL20, IL24), interferons (IFNĸ) and chemokines (CCL24, CCL26). The data have important implications for the recurrence of HPV-driven pathology following microwave treatment.

Plasma membrane gap junctions, formed by connexin proteins, are responsible for direct intercellular communication between adjacent cells by allowing the exchange of ions, metabolites, secondary messengers and microRNAs. Connexin 43 (Cx43) is a widely expressed gap junction protein. Cx43 mis-expression is associated with various disease states, including chronic non-healing wounds. Loss of Cx43 from the plasma membrane is important to allow wound resolution. Cx43 expression increases, and gap junctions reform, upon wound closure. Cx43 directly interacts with Dlg1, a membrane-associated guanylate kinase (MAGUK) protein, which controls cell shape and polarity. In this study, AlphaFold3 modelling predicted an interaction between amino acids 263–269 and 302–320 of Cx43 and the SH3, HOOK and GUK domains of Dlg1. Since Dlg1 is required for maintaining Cx43 protein levels at the plasma membrane, we hypothesised that Dlg1 may regulate wound healing via its functional interaction with Cx43. Cx43 and Dlg1 relocated to the cytoplasm of cells at the wound edge in scrape wound assays and co-located in pseudopodia of cells migrating towards the wound edge in live cell imaging experiments. Scratch wound assays carried out in HaCaT cells treated with siRNA targeting Dlg1 showed that Dlg1 depletion resulted in reduced wound closure rates. This was not due to changes in cell migration, suggesting that Dlg1 controlled cell proliferation. The data demonstrate that Cx43 and Dlg1 both contribute to wound closure, but that Dlg1 has other crucial functions in migrating cells separate from its role in maintaining Cx43 at the plasma membrane.

High risk human papillomaviruses (hrHPVs) cause most cervical cancers. Cervical screening programmes aim to identify precancerous disease using detection of hrHPV nucleic acid using clinician‐taken liquid‐based cytology (LBC) samples, or increasingly, self‐taken samples (STSs). However, STSs are incompatible with traditional cytology triage. Therefore, development of novel triage tests is essential. Quantification of cellular and viral mRNA biomarkers is one option, but little is known about mRNA quality in STSs. We extracted RNA from two sets of STSs (reflecting separate sampling devices) from the Scottish HPV Archive (SHA) and the Swedish Cervical Cytology Biobank (SCCB). We investigated whether the ACTB , GAPDH and p16 RNAs could be amplified by reverse transcription quantitative PCR (RT‐qPCR). RNA was degraded in both sets of samples, but samples from the Scottish HPV Archive were generally suitable for RT‐qPCR analysis, while samples from the SCCB were mostly unsuitable. Genomic DNA contamination was detected in 11.6% of samples. The quantity and quality of RNA derived from STSs was unaffected by storage of the original sample at −70°C for a period of 1 year. These data suggest that feasibility of utilising STSs for mRNA expression work is device‐dependent and that optimisation of collection and storage systems is warranted.

High risk human papillomaviruses (hrHPVs) cause most cervical cancers. Cervical screening programmes aim to identify precancerous disease using detection of hrHPV nucleic acid using clinician-taken liquid-based cytology (LBC) samples, or increasingly, self-taken samples (STSs). However, STSs are incompatible with traditional cytology triage. Therefore, development of novel triage tests is essential. Quantification of cellular and viral mRNA biomarkers is one option, but little is known about mRNA quality in STSs. We extracted RNA from two sets of STSs (reflecting separate sampling devices) from the Scottish HPV Archive (SHA) and the Swedish Cervical Cytology Biobank (SCCB). We investigated whether the ACTB , GAPDH and p16 RNAs could be amplified by reverse transcription quantitative PCR (RT-qPCR). RNA was degraded in both sets of samples, but samples from the Scottish HPV Archive were generally suitable for RT-qPCR analysis, while samples from the SCCB were mostly unsuitable. Genomic DNA contamination was detected in 11.6% of samples. The quantity and quality of RNA derived from STSs was unaffected by storage of the original sample at -70°C for a period of one year. These data suggest that feasibility of utilising STSs for mRNA expression work is device-dependent and that optimisation of collection and storage systems is warranted.

Human rhinoviruses (HRV) are a leading cause of the common cold but can often lead to respiratory complications such as wheeze in young children. In a transcriptomic study of respiratory nasal swab specimens from children hospitalised with acute wheeze, a significant alteration was found in the expression of the serine/arginine rich splicing factor (SRSF) kinase, SRPK1, between HRV positive children with acute exacerbations and HRV negative controls. As this kinase can regulate host RNA splicing, we hypothesised that HRV infection could dysregulate the expression of host mRNAs to affect antiviral functions or to alter the morphological features of the infected respiratory epithelium. Here, we show that pharmacological inhibition of SRPK1 in primary bronchial epithelial cells resulted in increased HRV16 replication while overexpression of SRPK1 reduced viral replication. In a primary bronchial epithelial 3D model infected with HRV16 decreased phosphorylation of SRSF1, 3 and 6 was observed. Furthermore, transcriptomic and alternative splicing (AS) bioinformatic analysis revealed the significantly altered AS of 1228 host genes during infection. Subsequent pathway analysis revealed the enrichment of most of these genes in networks related to cilia development and function. HRV16 infection led to significantly decreased cilia length and total cilia numbers in the primary bronchial epithelial 3D model together with changes to selected cilia proteins. Overall, this investigation has unravelled novel cellular networks implemented during HRV infection that may lead to acute exacerbations of respiratory infections. Author summary: Human rhinoviruses cause the common cold. In immunocompetent individuals this is usually a self-limiting infection. However, in young children and the elderly, infection can lead to complications such as bronchiolitis, croup, and wheezing. Rhinovirus infection can exacerbate chronic conditions such as cystic fibrosis, chronic obstructive pulmonary disease and asthma. Understanding the molecular pathology of this exacerbation could lead to new avenues for therapy. In this study, we discovered that a multifunctional cellular enzyme called serine arginine protein kinase 1 (SRPK1) is a restriction factor for human rhinovirus 16 (HRV16) infection. One key cellular function of SRPK1 is to regulate RNA splicing through modifying the SR proteins that normally enhance splicing. In three dimensional tissues grown from human bronchial epithelial cells, we found that HRV16 infection led to decreased levels of modified SR proteins. This change resulted in significant alterations in RNA expression in the infected cell. Most of these alterations affected production of the correct versions of cilia proteins resulting in reduced cilia numbers and cilia blunting. This type of damage due to HRV infection would result in inefficient clearance of subsequent viral infections prolonging the viral infection leading to lower respiratory tract infection and to exacerbations of existing chronic disease.

Human papillomaviruses (HPV) infect epithelia to cause benign lesions or warts. However, the so-called “high risk” HPVs infecting the anogenital region and the oropharynx can cause precancerous lesions that may progress to malignant tumours. Understanding the HPV life cycle is important to the discovery of novel antiviral therapies. HPV uses cellular splicing to produce the full suite of viral mRNAs. Members of the serine/arginine rich (SR) protein family can positively regulate splicing. SR protein activity and cellular location is regulated by phosphorylation of their serine-arginine domains. SR protein kinases (SRPK) can phosphorylate SR proteins. This licenses their nuclear entry and promotes nuclear splicing together with another SR protein kinase, Clk1. SRPIN340 is a specific inhibitor of SRPK1. It has been reported to inhibit replication of HCV, Sindbis virus and HIV. We show here that SRPIN340 inhibits the expression of the viral replication and transcription factor, E2. Loss of HPV E4 and L1 late proteins was also observed. RNA sequencing showed SRPIN340 treatment resulted in gene expression changes opposite to those induced by HPV16 infection. In particular, the loss of the epithelial barrier was restored. SRPIN340 treatment led to changes in alternative splicing of 935 RNAs and pathway analysis showed a predominance of changes to RNAs encoding proteins involved in chromatin conformation, DNA repair and RNA processing. Short term SRPIN340 treatment (two to three days) was not associated with changes in proliferation or differentiation of keratinocytes. Since SRPK1 controls the E2 viral replication and transcription factor, targeting this kinase, or the phosphorylation events it mediates, could be considered as a therapeutic strategy for HPV16 infection.

Human papillomaviruses (HPV) infect epithelial causing benign lesions. However, the so-called “high risk” HPVs that infect the anogenital regions and the oropharynx can cause precancerous lesions that can progress to malignant tumours. Understanding the HPV life cycle is important to the discovery of novel antiviral therapies. HPV uses cellular splicing for the production of the full suite of viral mRNAs. Members of the serine/arginine rich (SR) protein family can positively regulate splicing. SR protein activity and cellular location is regulated by kinases through phosphorylation of their serine-arginine domains. SR protein kinase 1 (SRPK1) phosphorylates SR proteins to licence their nuclear entry and can promote nuclear splicing in conjunction with another SR protein kinase, Clk1. SRPIN340 is a specific SRPK1 inhibitor that has been reported to inhibit replication of HCV, Sindbis virus and HIV. Using organotypic raft cultures supporting the HPV16 life cycle, we show that SRPIN340 inhibits the expression of the viral replication and transcription factor, E2. Drug-mediated loss of E2 is specifically associated with inhibition of late gene expression since early gene expression is unaffected. RNA sequencing revealed that SRPIN340 treatment resulted in many gene expression changes opposite to those induced by HPV16 infection. In particular, the loss of epithelial barrier structure and immune function was restored. SRPIN340 treatment resulted in changes in alternative splicing of 935 RNAs and pathway analysis showed a predominance of changes to RNAs encoding proteins involved in chromatin conformation, DNA repair and RNA processing. SRPIN340 treatment was not associated with changes in proliferation or differentiation of keratinocytes. Collectively, these results provide evidence that SRPK1 is a host factor that is essential for HPV16 replication and therefore, targeting this factor, or the phosphorylation events it mediates, could be considered as a therapeutic strategy for HPV16 infection. AUTHOR SUMMARY: Human papillomavirus (HPV) can cause precancerous and cancerous tumours of the anogenital region, for example, the cervix. HPV infects epithelial cells and utilizes the host cell gene expression machinery to produce its own proteins. Splicing is a key mechanism used by HPV to synthesise messenger RNAs that encode the viral proteins. Splicing is positively controlled by SR proteins which require to be phosphorylated by SR protein kinase (SRPK) to enter the nucleus and regulate splicing. SRPIN340 is a specific inhibitor of SRPK. SRPIN340 treatment of HPV16-infected epithelial cells resulted in loss of expression of the viral replication and transcription factor, E2 and abrogation of late events in the viral life cycle. SRPIN340 treatment counteracted some gene expression changes to the epithelium known to be cause by HPV infection. These data suggest that SRPK1 is essential for the HPV life cycle and that it could be a potential anti-viral target.

Human papillomaviruses (HPVs) infect cutaneous and mucosal epithelia to cause benign (warts) and malignant lesions (e.g. cervical cancer). Bovine papillomaviruses (BPVs) infect fibroblasts to cause fibropapillomas but can also infect cutaneous epithelial cells. For HPV-1, -16, -31 and BPV-1, cis-acting RNA elements in the late 3' untranslated region (3'UTR) control expression of virus proteins by binding host cell proteins. The present study compared the effects on gene expression of the cis-acting elements of seven PV late 3'UTRs (HPV-6b, -11, -16, -31 and BPV-1, -3 and -4) representing a range of different genera and species and pathological properties. pSV-beta-galactosidase reporter plasmids containing the late 3'UTRs from seven PVs were transiently transfected into cervical adenocarcinoma HeLa cells, and reporter gene expression quantified by reverse transcription-quantitative PCR and a beta-galactosidase assay. All elements inhibited gene expression in keratinocytes. Cancer-related types HPV-16 and -31, had the greatest inhibitory activity whereas the lowest inhibition was found in the non-cancer related types, BPV-3 and HPV-11. Using RBPmap version 1.1, bioinformatics predictions of factors binding the elements identified proteins which function mainly in mRNA splicing. Markedly, in terms of protein binding motifs, BPV late 3'UTR elements were similar to those of HPV-1a but not to other HPVs. Using HPV-1a as a model and siRNA depletion, the bioinformatics predictions were tested and it was found that PABPC4 was responsible for some of the 3'UTR repressive activity. The data revealed candidate proteins that could control PV late gene expression.

Papillomavirus gene regulation is largely post-transcriptional due to overlapping open reading frames and the use of alternative polyadenylation and alternative splicing to produce the full suite of viral mRNAs. These processes are controlled by a wide range of cellular RNA binding proteins (RPBs), including constitutive splicing factors and cleavage and polyadenylation machinery, but also factors that regulate these processes, for example, SR and hnRNP proteins. Like cellular RNAs, papillomavirus RNAs have been shown to bind many such proteins. The life cycle of papillomaviruses is intimately linked to differentiation of the epithelial tissues the virus infects. For example, viral late mRNAs and proteins are expressed only in the most differentiated epithelial layers to avoid recognition by the host immune response. Papillomavirus genome replication is linked to the DNA damage response and viral chromatin conformation, processes which also link to RNA processing. Challenges with respect to elucidating how RBPs regulate the viral life cycle include consideration of the orchestrated spatial aspect of viral gene expression in an infected epithelium and the epigenetic nature of the viral episomal genome. This review discusses RBPs that control viral gene expression, and how the connectivity of various nuclear processes might contribute to viral mRNA production.