The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCEHuman papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4^L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and the production of new virions.
Gap junctions, composed of Cxs (connexins), allow direct intercellular communication. Gap junctions are often lost during the development of malignancy, although the processes behind this are not fully understood. Cx43 is a widely expressed Cx with a long cytoplasmic C-terminal tail that contains several potential protein-interaction domains. Previously, in a model of cervical carcinogenesis, we showed that the loss of gap junctional communication correlated with relocalization of Cx43 to the cytoplasm late in tumorigenesis. In the present study, we demonstrate a similar pattern of altered expression for the hDlg (human discs large) MAGUK (membrane-associated guanylate kinase) family tumour suppressor protein in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg. hDlg is not required for Cx43 internalization in W12GPXY cells. Instead, hDlg appears to have a role in maintaining a cytoplasmic pool of Cx43. These results demonstrate that hDlg is a physiologically relevant regulator of Cx43 in transformed epithelial cells.
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.
Persistent infection with cancer risk-related viruses leads to molecular, cellular and immune response changes in host organisms that in some cases direct cellular transformation. Alternative splicing is a conserved cellular process that increases the coding complexity of genomes at the pre-mRNA processing stage. Human and other animal tumour viruses use alternative splicing as a process to maximize their transcriptomes and proteomes. Medical therapeutics to clear persistent viral infections are still limited. However, specific lessons learned in some viruses [e.g. HIV and HCV (hepatitis C virus)] suggest that drug-directed inhibition of alternative splicing could be useful for this purpose. The present review describes the basic mechanisms of constitutive and alternative splicing in a cellular context and known splicing patterns and the mechanisms by which these might be achieved for the major human infective tumour viruses. The roles of splicing-related proteins expressed by these viruses in cellular and viral gene regulation are explored. Moreover, we discuss some currently available drugs targeting SR (serine/arginine-rich) proteins that are the main regulators of constitutive and alternative splicing, and their potential use in treatment for so-called persistent viral infections.
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