Search PubMed⌕ Search

Biomedical subjects

B L Semler

Publications and source records attributed to B L Semler.

At least 55 records · Page 3Linked to original sources
PubMed1991

Poliovirus translation initiation: differential effects of directed and selected mutations in the 5' noncoding region of viral RNAs.

We have analyzed the translational defects of a number of mutations in the 5' noncoding region of poliovirus type 1 RNA. These mutations fall into three categories: (1) two mutations which resulted in temperature sensitive (ts) viruses, (2) the second-site mutations responsible for the reversion of the two ts viruses, and (3) mutations which were lethal to virus production. RNAs containing either of the ts mutations translated in vitro at levels significantly lower than wild-type levels. RNAs containing the respective second-site reversions had corrected these translational defects to levels corresponding to their viral growth potentials. Unlike in vitro translation of wild-type poliovirus RNA, translation of the RNAs which gave rise to ts mutant viruses was not stimulated by the addition of an S10 fraction from an uninfected HeLa cell extract to a rabbit reticulocyte lysate (RRL). In vitro translation of the mutant RNAs (corresponding to the ts viruses) in a RRL was stimulated by factors present in a ribosomal salt wash (RSW) from a HeLa extract, although the levels of stimulation were only half those seen for wild-type. These results suggest that the stimulatory factors present in the RSW have a decreased affinity for the mutant RNA templates but can, to some extent interact, with such RNAs if provided in high enough concentration. The in vitro translation of RNAs containing either of the lethal mutations was not stimulated by factors present in the S10 or the RSW. Taken together, our data suggest a correlation between the ability of a genetically altered RNA to respond to translation stimulatory factors in vitro and the ability of that mutation to be recovered in infectious virus. In addition, we have identified the in vivo-selected reversion of translational defects for two different ts viruses.

DNA Mutational Analysis↗
PubMed1991

Self-cleaving proteases.

Research on the activity of self-cleaving proteases in bacterial, mammalian and virus-infected cells is reviewed, with an emphasis on the diversity of regulatory systems controlled by protein processing. Each of these three groups will be considered in turn by focusing on the following systems: the Rec A-dependent intramolecular cleavage of the Escherichia coli SOS response protein, LexA; the intramolecular activation of the mammalian aspartic acid protease, pepsinogen; and the autocatalytic cleavage of polyproteins synthesized by picornaviruses.

Bacterial Proteins↗
PubMed1991

Role for the P4 amino acid residue in substrate utilization by the poliovirus 3CD proteinase.

Amino acid insertions or substitutions were introduced into the poliovirus P1 capsid precursor at locations proximal to the two known Q-G cleavage sites to examine the role of the P4 residue in substrate processing by proteinase 3CD. Analysis of the processing profile of P1 precursors containing four-amino-acid insertions into the carboxy terminus of VP3 or a single-amino-acid substitution at the P4 position of the VP3-VP1 cleavage site demonstrates that substitution of the alanine residue in the P4 position of the VP3-VP1 cleavage site significantly affects cleavage at that site by proteinase 3CD. A single-amino-acid substitution at the P4 position of the VP0-VP3 cleavage site, on the other hand, has only a slight effect on 3CD-mediated processing at this cleavage site. Finally, analysis of six amino acid insertion mutations containing Q-G amino acid pairs demonstrates that the in vitro and in vivo selection of a cleavage site from two adjacent Q-G amino acid pairs depends on the presence of an alanine in the P4 position of the cleaved site. Our data provide genetic and biochemical evidence that the alanine residue in the P4 position of the VP3-VP1 cleavage site is a required substrate determinant for the recognition and cleavage of that site by proteinase 3CD and suggest that the P4 alanine residue may be specifically recognized by proteinase 3CD.

3C Viral Proteases↗
PubMed1991

Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis.

A molecular genetic analysis has been combined with an in vitro biochemical approach to define the functional interactions required for nucleotidyl protein formation during poliovirus RNA synthesis. A site-directed lesion into the hydrophobic domain of a viral membrane protein produced a mutant virus that is defective in RNA synthesis at 39 degrees C. The phenotypic expression of this lesion affects initiation of RNA synthesis, in vitro uridylylation of the genome-linked protein (VPg), and the in vivo synthesis of plus-strand viral RNAs. Our results support a model that employs a viral membrane protein as carrier for VPg in the initiation of plus-strand RNA synthesis. Our data also suggest that a separate mechanism could be used in the initiation of minus-strand RNA synthesis, thereby providing a means for strand-specific regulation of picornavirus RNA replication.

Amino Acid Sequence↗
PubMed1991

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.

Capsid↗
PubMed1990

Species-specific substrate interaction of picornavirus 3C proteinase suballelic exchange mutants.

The substrate recognition properties of the polio-virus type 1 and coxsackievirus B3 3C proteinases have been examined in vitro by allelic and suballelic exchange of 3C between the cloned virus genomes. The activity of the altered 3C proteinases was examined by translation of synthetic RNA in a rabbit reticulocyte lysate/HeLa cell extract translation system. Analysis of the subsequent processing of virus polyproteins by the altered 3C proteinases showed that all of the mutant proteinases maintained some catalytic activity. The disruption of polyprotein cleavages mediated by 3C followed a distinct pattern, suggesting a specific order of events in processing the polyprotein. Differences in cleavage activity of mutant proteinases when tested on coxsackievirus or poliovirus protein substrates suggest that, although structural elements throughout the proteinase play a role in efficient substrate utilization, the carboxyl-terminal region of the 3C proteinase contains elements most important in species-specific substrate recognition.

Alleles↗
PubMed1990

A genetic locus in mutant poliovirus genomes involved in overproduction of RNA polymerase and 3C proteinase.

A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.

3C Viral Proteases↗
PubMed1990

Neurovirulence determinants of genetically engineered Theiler viruses.

Theiler murine encephalomyelitis viruses (TMEVs) are picornaviruses that cause enteric and neurological disease in mice. The GDVII strain and other members of the GDVII subgroup are highly virulent and cause an acute, fatal polioencephalomyelitis following intracerebral inoculation, whereas the DA stain and other members of the TO subgroup cause a persistent, demyelinating infection. We previously produced a full-length, infectious DA cDNA clone. We now describe the generation of a full-length, infectious GDVII cDNA clone and the subsequent production of intratypic chimeric cDNAs and intratypic recombinant viruses. Inoculation of the recombinant viruses into mice demonstrated that a major determinant of TMEV neurovirulence is within the GDVII 1B (capsid protein VP2)-2C coding region, most likely in the GDVII 1B (VP2)-2A coding region. Genomic sequences 5' to this region of GDVII RNA also contribute to expression of the full neurovirulence phenotype. These data demonstrate the multigenic nature of TMEV neurovirulence, as has been reported for other viruses.

Amino Acid Sequence↗
PubMed1990

A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation.

A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.

Amino Acid Sequence↗
PubMed1989

Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage.

A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.

Amino Acid Sequence↗
PubMed1989

Polyprotein processing of Theiler's murine encephalomyelitis virus.

To investigate polyprotein processing of Theiler's murine encephalomyelitis viruses, we analyzed in vitro translation reactions programmed by in vitro-derived transcripts from an infectious full-length cDNA clone of the DA strain of Theiler's virus. To help identify the proteinases that carried out the processing, we modified the DA cDNA clone transcription template by linearization with different restriction endonucleases that generate templates of different lengths or by constructing linker insertion or deletion mutations or both in putative proteinase-coding regions. Protein 3C carried out most of the cleavages of the polyprotein, as is true for the other picornaviruses that have been studied. A second proteinase also appeared active at the LP12A-2B junction. A protein of slightly faster mobility than the leader protein was seen with translation of transcripts derived from DA cDNA but not GDVII cDNA. This protein may be synthesized from an alternative initiation site in the DA leader-coding region out of phase with the polyprotein reading frame. Our findings are relevant to ongoing investigations of the abnormal virus expression seen in DA virus late demyelinating disease, since polyprotein processing is critical in regulating picornaviral gene expression.

Animals↗
PubMed1989

Infectious cDNA clones of the DA strain of Theiler's murine encephalomyelitis virus.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis viruses cause a persistent demyelinating infection, whereas the GDVII strain and other GDVII subgroup strains cause an acute lethal polioencephalomyelitis. We generated an infectious DA cDNA clone inserted into a transcription vector. Virus derived from transfection of transcripts produced a demyelinating disease indistinguishable from that of wild-type virus. The infectious clone provides a critical reagent for the production of interstrain recombinant viruses to help identify genetic loci responsible for the biological activities of the strains.

Animals↗
PubMed1989

The deletion of 41 proximal nucleotides reverts a poliovirus mutant containing a temperature-sensitive lesion in the 5' noncoding region of genomic RNA.

We generated a number of small deletions and insertions in the 5' noncoding region of an infectious cDNA copy of the poliovirus RNA genome. Transfection of these mutated cDNAs into COS-1 cells produced the following phenotypic categories: (i) wild-type mutations, (ii) lethal mutations, (iii) mutations exhibiting slow growth or low-titer properties, and (iv) temperature-sensitive (ts) mutations. The deletion of nucleotides 221 to 224 produced a ts virus, 220D1. Mutant 220D1 was found to have a dramatic reduction in growth, virus-specific protein and RNA synthesis, and the shutoff of host cell protein synthesis at 37 or 39 degrees C compared with 33 degrees C. Temperature shift experiments showed that the mutant viral RNA is not an effective template for protein or RNA synthesis at 39 degrees C and suggested a decreased stability of the 220D1 RNA at 39 degrees C. Selection for a non-ts revertant of 220D1 yielded the virus R2, which was no longer ts for growth or viral protein and RNA synthesis. Sequencing the 5' noncoding region of the genomic RNA from R2 revealed the deletion of 41 proximal nucleotides for an overall deletion of nucleotides 184 to 228. These data suggest that the deleted sequences are nonessential to the poliovirus life cycle during growth in HeLa cells. According to computer-predicted RNA secondary structures of the 5' noncoding region of poliovirus RNA, the R2 revertant virus has deleted an entire predicted stem-loop structure.

Animals↗
PubMed1989

Chimeric picornavirus polyproteins demonstrate a common 3C proteinase substrate specificity.

Cross-species proteolytic processing was demonstrated by the 3C proteinases of human rhinovirus 14 and coxsackievirus B3 on poliovirus-specific polypeptide precursors. Chimeric picornavirus cDNA genomes were constructed in a T7 transcription vector in which the poliovirus 3C coding region was substituted with the corresponding allele from human rhinovirus 14 or coxsackievirus B3. In vitro translation and processing of the polypeptides encoded by the chimeric genomes demonstrated that the proteolytic processing of poliovirus P2 region (nonstructural) proteins could be functionally substituted by the heterologous proteinases. In contrast, the 3C proteinase activities expressed from the chimeric genomes were incapable of recognizing the poliovirus-specific processing sites within the capsid precursor. Since the amino acid sequences flanking and inclusive of the P2 region cleavage sites of the three viruses are not stringently conserved, these results provide evidence for the existence of common conformational determinants necessary for 3C-mediated processing.

Chimera↗
PubMed1988

Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs.

The processing of poliovirus precursor polypeptides provides a valuable system in which to study the recognition and interaction of a proteolytic enzyme with its substrates. Processing of the poliovirus polyprotein includes cleavage between 9 of 13 available glutamineglycine (Q-G) pairs by the activity of a virally encoded proteinase, 3C. In this study, we assess the importance of primary, secondary, and tertiary structural determinants in the cleavage at two Q-G pairs in the capsid protein precursor, P1. Employing site-directed mutagenesis of cDNA copies of poliovirus RNA, we have made specific alterations in regions of the P1 capsid precursor and have assayed the effect of these alterations on proteinase cleavage at the two Q-G pairs. We have also introduced additional Q-G pairs into P1 and demonstrated that the proteinase can recognize some of the inserted Q-G pairs as cleavage sites. By correlating the predicted three-dimensional structures and the processing phenotypes of several altered P1 precursors, we are able to rank the importance of determinants required for P1 processing. While a Q-G pair appears to be the primary determinant in proteinase recognition, the tertiary location of a Q-G pair in the precursor either allows or prevents processing at that pair. Our results also suggest that the proper folding of at least two of the three P1 beta-barrel structures is required for efficient proteinase cleavage at Q-G pairs.

Amino Acid Sequence↗
PubMed1988

Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor.

The rate and extent of polyprotein processing are the major steps controlling picornavirus gene expression. It is, therefore, important to determine the enzymes responsible for each proteolytic event. The poliovirus protein 3C has been identified as a proteinase which specifically cleaves between Q-G pairs. However, recent data have suggested that 3C precursor polypeptides containing 3C sequences may also have proteolytic capabilities. In this study we have analyzed the cleavage specificities of protein 3C and its precursor, 3CD. We have carried out in vitro translation of genetically altered poliovirus mRNAs to demonstrate that 3CD is required for efficient processing of the P1 capsid precursor to capsid proteins. In addition, we suggest 3CD and 3C process Q-G pairs in the P2 and P3 precursors with similar efficiencies.

Capsid↗
PubMed1988

Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5'-noncoding regions of viral RNAs.

We describe the isolation of a variant of a polio--coxsackie recombinant virus (PCV110) containing a genomic RNA with a chimeric 5'-noncoding region. The variant virus [designated PCV110(1)] has growth and biosynthetic properties that are quite different from the original, temperature-sensitive isolate of the recombinant virus [designated PCV110(4)]. Nucleotide sequencing of the 5'-noncoding region of RNA from PCV110(1) revealed a 4-base deletion within the substituted coxsackievirus region of the chimeric genome that may contribute to the loss of temperature sensitivity of this variant recombinant virus. In addition, we have generated new recombinant viruses that contain (1) coxsackievirus sequences within the N66-N627 region of the poliovirus genome and (2) coxsackievirus sequences substituted from N1-N627 in the poliovirus genome. These recombinant viruses are not temperature sensitive for growth at 37 degrees and have biosynthetic properties similar to those of wild-type poliovirus. Our results provide evidence that replicase recognition signals encoded in the 5' noncoding regions of enterovirus genomic RNAs are not strictly sequence specific.

Base Sequence↗