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Bert L Semler

Publications and source records attributed to Bert L Semler.

17 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2005

Atomic force microscopy analysis of icosahedral virus RNA.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

Bromovirus↗
PubMed2005

Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase.

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

Allosteric Regulation↗
PubMed2005

An authentic 3' noncoding region is necessary for efficient poliovirus replication.

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

3' Untranslated Regions↗
PubMed2005

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

3C Viral Proteases↗
PubMed2005

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1.

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.

5' Untranslated Regions↗
PubMed2004

Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA.

The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.

5' Flanking Region↗
PubMed2004

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES.

The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.

DNA-Binding Proteins↗
PubMed2004

Regulation of picornavirus gene expression.

Members of the Picornaviridae are positive- strand RNA viruses that cause a variety of human diseases such as poliomyelitis, the common cold, myocarditis, and hepatitis. Although the diseases caused by picornaviruses are diverse, the genome organization and mechanisms of gene expression are highly conserved among family members. This review will discuss the mechanisms of viral gene expression including cap-independent translation initiation, host cell translation shut off, viral polyprotein processing, and RNA replication.

Gene Expression Regulation, Viral↗
PubMed2004

Differential rescue of poliovirus RNA replication functions by genetically modified RNA polymerase precursors.

We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

5' Untranslated Regions↗
PubMed2004

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis.

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.

3' Untranslated Regions↗
PubMed2004

Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus.

The viral RNA-dependent RNA polymerase (3D(pol)) is highly conserved between the closely related enteroviruses poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3). In this study, we generated PV1/CVB3 chimeric polymerase sequences in the context of full-length poliovirus transcripts to determine the role of different subdomains within the RNA-dependent RNA polymerase of PV1 that are required for functions critical for RNA replication in vitro and in cell culture. The substitution of CVB3 sequences in the carboxy-terminal portion (thumb subdomain) of the polymerase resulted in transcripts incapable of RNA replication. In contrast, three of the seven chimeras were capable of synthesizing RNA, albeit to reduced levels compared to that of wild-type PV1 RNA. Interestingly, one of the replication-competent chimeras (CPP) displayed an inability to generate positive strands, indicating the presence of amino-terminal sequences within the 3D polymerase and/or the 3D domain of the 3CD precursor polypeptide that are necessary for the assembly of strand-specific RNA synthesis complexes. In some constructs, the partial reestablishment of PV1 amino acid sequences in this region was capable of rescuing RNA replication in vitro and in cell culture.

3C Viral Proteases↗
PubMed2004

Poliovirus proves IRES-istible in vivo.

The genetic basis for the attenuation of polio vaccines has been known since the 1980s. Changes in the internal ribosome entry site, within the 5' noncoding region of genomic RNAs, were presumed to reduce translation in certain target organs, leading to the conclusion that attenuation is mediated at the level of translation. A report in this issue of the JCI reveals that poliovirus tropism is, in part, determined after internal ribosome entry.

5' Untranslated Regions↗
PubMed2003

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus.

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.

Amino Acid Sequence↗
PubMed2002

Subdomain specific functions of the RNA polymerase region of poliovirus 3CD polypeptide.

The 3D polymerase domain of the poliovirus 3CD polypeptide plays a role in modulating its RNA binding and protein processing activities, even though the proteinase catalytic site and RNA binding determinants appear to reside within the 3C(pro) portion of the molecule. In this study, we have generated recombinant 3CD polypeptides that contain chimeric 3D polymerase domains representing suballelic sequence exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) to determine which portions of the 3D domain are responsible for influencing these activities. By utilizing these recombinant protein chimeras in protein processing and RNA binding studies in vitro, we have generated data suggesting the presence of separate subdomains within the polymerase domain of 3CD that may independently modulate its RNA binding and protein processing activities. In predicting where our sequence exchanges map by utilizing the previously published three-dimensional structure of the PV1 3D polymerase, we present evidence that sequences contained within the RNA recognition motif of the polymerase are critical for 3CD function in recognizing the 5' RNA cloverleaf. Furthermore, our protein processing data indicate that at least some of the substrate recognition and processing determinants within the 3D domain of 3CD are separate and distinct from the RNA binding determinants in this domain.

3C Viral Proteases↗
PubMed2002

Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication.

The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

Amino Acid Sequence↗