Re-localization of Cellular Protein SRp20 during Poliovirus Infection: Bridging a Viral IRES to the Host Cell Translation Apparatus

Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions

Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed “unlinkase,” that recognizes and cleaves the unique 5′ tyrosyl-RNA phosphodiester bond found at the 5′ end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate

Integration of the yeast retrovirus-like element Ty3 upstream of a human tRNA gene expressed in yeast.

The retrovirus-like element Ty3 of Saccharomyces cerevisae integrates into the yeast genomic DNA in a position specific manner. Ty3 integrates within 1-2 base pairs of the site of transcription initiation by RNA polymerase III. The human tRNA(Lys)3 gene was used as a target for transposition in a plasmid-based assay to determine whether Ty3 integration can be targeted to a human tRNA gene. Each transposition event observed was adjacent to the site of initiation of transcription of the human tRNA gene. Therefore, heterologous tRNA genes can serve as targets for Ty3 in yeast. This is a first step toward development of a system for targeted integrations in heterologous organisms

A chimeric Ty3/Moloney murine leukemia virus integrase protein is active in vivo.

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo(r)-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains

A chimeric Ty3/Moloney murine leukemia virus integrase protein is active in vivo.

This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neo(r)-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains

A truncation mutant of the 95-kilodalton subunit of transcription factor IIIC reveals asymmetry in Ty3 integration.

Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathione S-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo

A truncation mutant of the 95-kilodalton subunit of transcription factor IIIC reveals asymmetry in Ty3 integration.

Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathione S-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo

A yeast sigma composite element, TY3, has properties of a retrotransposon.

Sigma is a 340- or 341-base pair repetitive element which is located almost exclusively within 19 base pairs of the 5' ends of various tRNA genes in the Saccharomyces cerevisiae genome. Although most sigma elements characterized to date are isolated insertions, a few of the elements occur relatively closely spaced. One of these pairs is a direct repeat of the sigma element separated by an internal domain 4.7 kilobase pairs in length. Not only does this structure resemble a composite transposable element, but regions within the sigma elements and intervening domain are homologous to conserved regions in retroviruses and retrotransposons of yeast and other organisms. Two features suggest that the sigma elements and intervening DNA transposed in a concerted event: only one of the two sigma elements is associated with a tRNA gene, and only the outside ends of the two elements are flanked by the 5-base pair direct repeats that usually flank individual sigma insertions. Examination of genomic DNA from five laboratory strains indicates that the 4.7 kilobase pair internal domain is present in one to four copies per haploid genome and that the genomic location of this domain differs from strain to strain. In addition, Northern blot analysis showed the presence of a 5.2 kilobase poly(A) transcript which hybridizes to both sigma and internal domain-specific probes. The existence of this composite element may suggest new ways to consider the mechanisms by which retrotransposons select their targets

A yeast sigma composite element, TY3, has properties of a retrotransposon.

Sigma is a 340- or 341-base pair repetitive element which is located almost exclusively within 19 base pairs of the 5' ends of various tRNA genes in the Saccharomyces cerevisiae genome. Although most sigma elements characterized to date are isolated insertions, a few of the elements occur relatively closely spaced. One of these pairs is a direct repeat of the sigma element separated by an internal domain 4.7 kilobase pairs in length. Not only does this structure resemble a composite transposable element, but regions within the sigma elements and intervening domain are homologous to conserved regions in retroviruses and retrotransposons of yeast and other organisms. Two features suggest that the sigma elements and intervening DNA transposed in a concerted event: only one of the two sigma elements is associated with a tRNA gene, and only the outside ends of the two elements are flanked by the 5-base pair direct repeats that usually flank individual sigma insertions. Examination of genomic DNA from five laboratory strains indicates that the 4.7 kilobase pair internal domain is present in one to four copies per haploid genome and that the genomic location of this domain differs from strain to strain. In addition, Northern blot analysis showed the presence of a 5.2 kilobase poly(A) transcript which hybridizes to both sigma and internal domain-specific probes. The existence of this composite element may suggest new ways to consider the mechanisms by which retrotransposons select their targets

A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation

A significant number of viral and cellular mRNAs utilize cap-independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap-independent translation requires noncanonical, cellular RNA-binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo-cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)-mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA-binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES-mediated translation), further documenting the multifunctional nature of this important class of cellular RNA-binding proteins