Our research focuses on how picornaviruses (including human rhinovirus, poliovirus, and coxsackievirus) regulate the expression and replication of their genomic RNAs in infected human cells. The limited coding capacity of picornavirus genomic RNAs results in a genetically-challenged RNA virus. As a result, picornaviruses have evolved to utilize host cell proteins in different steps of their intracellular, cytoplasmic replication cycles. Somewhat surprisingly, several proteins known or suspected to have roles in viral gene expression and RNA replication reside primarily in the nucleus of uninfected mammalian cells. Due to viral-specific alteration of protein trafficking between the cytoplasm and the nucleus of infected cells, such proteins become available for viral functions in the cytoplasm.
We have identified two HeLa cell proteins that fall into this unusual category of nuclear proteins involved in the cytoplasmic functions of poliovirus. The first of these proteins is SRp20, a member of a group of pre-mRNA splicing factors known to act at several steps during constitutive eukaryotic mRNA splicing. SRp20 has also been implicated in the export of mRNAs from the nucleus to the cytoplasm. Using in vitro translation assays as well as RNA interference experiments in HeLa cells, we demonstrated that SRp20 is required for poliovirus translation initiation. We have used confocal microscopy to show that within 3 hr post-infection, SRp20 begins to re-localize from the nucleus to the cytoplasm in poliovirus-infected neuroblastoma cells. Some of this cytoplasmic SRp20 co-localizes with another host cell RNA binding protein (PCBP2) that is involved in poliovirus IRES-mediated translation initiation. We generated an SRp20 deletion construct (SRp20deltaRRM) that lacks the RNA-recognition motif but still contains the domain required for interaction with PCBP2 (the RS domain). We found that the localization of this mutated protein is similar to wild type SRp20 in mock- or poliovirus-infected cells, and SRp20deltaRRM also partially co-localizes with PCBP2 in infected cells at 3 hr post-infection. In addition, we showed that expression of SRp20deltaRRM results in an approximate two-log defect in poliovirus growth, suggesting that the deleted protein acts as a dominant negative factor in poliovirus translation.
A second predominantly nuclear protein that re-localizes to the cytoplasm of cells infected with poliovirus is hnRNP C, a highly abundant protein in human cells that functions in cellular mRNA biogenesis processes in the nucleus, including mRNA splicing and stabilization of pre-mRNA. We found that hnRNP C binds to both the 5’ and 3’ ends of poliovirus negative-strand RNAs, suggesting a possible role for these RNP complexes in positive-strand viral RNA synthesis. We used retrovirus-mediated expression of hnRNP C-specific short hairpin RNAs (shRNAs) to reduce the levels of hnRNP C proteins in HeLa cells. Poliovirus yields were decreased five-fold in hnRNP C-specific shRNA-treated cells compared to infections of control shRNA-treated cells. Quantitative real-time PCR analysis revealed that the accumulation of positive-strand RNA was selectively decreased in hnRNP C-depleted cells during the early phases of poliovirus infection. Our results suggest that cellular hnRNP C proteins play an important role in the formation of positive-strand RNA replication complexes in infected cells. In addition to generating mechanistic insights into picornavirus-host cell interactions, results from our studies should identify molecular targets for antiviral therapies as well as the nature of specific macromolecular interactions that regulate viral gene expression.
Another research focus of Dr. Semler’s laboratory stems from his interest in IRES-mediated translation initiation on viral mRNAs. This mode of translation is also utilized by a limited number of cellular mRNAs whose functions may be required when normal cap-dependent translation is shut down (e.g., during mitosis or in cells undergoing physiological stress). One such mRNA encodes a murine voltage-gated potassium channel, Kv1.4. Potassium channels are known to regulate ion balance, membrane potential, secretion, and cell excitability. The specific mRNA for Kv1.4 is expressed in brain, heart, and skeletal muscle, and there appears to be translational regulation of its expression. They have previously shown that the long (1.2 kb) 5’ noncoding region of Kv1.4 mRNA contains an IRES element that is capable of directing translation initiation. Their research is currently focused on identification of cellular RNA binding proteins that interact with the Kv1.4 IRES and allow this mRNA to bypass the normal requirements for cap-dependent initiation via ribosome scanning. Data from this work should provide new insights into the mechanisms of internal ribosome entry used by specific viral and cellular mRNAs, mechanisms that will ultimately be crucial to understanding of cellular growth control (and its loss in transformed cells) since key cellular regulatory proteins like c-myc, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) are all produced from mRNAs harboring IRES elements in their 5’ noncoding regions.