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B L Semler

Publications and source records attributed to B L Semler.

At least 19 recordsLinked to original sources
PubMed1992

High frequency of single-base transitions and extreme frequency of precise multiple-base reversion mutations in poliovirus.

We employed independent clones of a temperature-sensitive mutant of type 1 poliovirus, 3AB-310/4, to quantitate the frequency of specific U----C transitions at nucleotide 5310, within the genomic region encoding polypeptide 3AB, which is involved in the initiation of RNA replication. Only this U----C base substitution restores the wild-type phenotypic ability to form plaques at 39 degrees C; the other two base substitutions at this site are lethal. The observed frequency of this specific transition averaged 2 x 10(-5), and all revertant viruses forming plaques at 39 degrees C contained the expected cytidine at nucleotide 5310. Incredibly, only 3 of 10 revertants exhibited this one specific U----C transition whereas 7 of 10 exhibited this same transition plus four additional base substitutions that precisely reverted temperature-sensitive 3AB-310/4 to wild-type poliovirus sequence (these latter four mutations had been introduced into 3AB-310/4 as silent third base mutations to provide new restriction sites in infectious cDNAs). No other mutations were detected in this polypeptide 3AB domain in either the single-base or the precise 5-base revertants. No intermediates were seen; all revertants exhibited either the single U----C transition at nucleotide 5310 or the same transition plus four precise reversions to the wild-type sequence at sites 8, 11, 43, and 46 bases distant from nucleotide 5310. Similar results were obtained after transfection of cDNA-derived transcripts. We discuss possible mechanisms for our data. These include (but may not be limited to) error-prone polymerase activity, sequential RNA recombination events joining independent mutations, or some unusual RNA editing process.

Base Sequence↗
PubMed1992

Alternate poliovirus nonstructural protein processing cascades generated by primary sites of 3C proteinase cleavage.

The post-translational regulation of picornavirus gene expression mediated by the cascade processing of viral proteins is not well understood. Both pulse-chase studies of infected cells and in vitro studies of the translation of poliovirus type 1 RNA transcribed from genomic cDNA clones indicate a specific cascade of polyprotein processing in which the P1, P2, and P3 precursor proteins are primary products of viral proteinase cleavage. We report the results of a short-time kinetic analysis of poliovirus type 1 protein processing in an in vitro translation system and in infected HeLa cells which indicate the existence of another, rapid pathway of polyprotein processing mediated by the activity of the 3C proteinase. The observed pathway is distinct from and in addition to the one previously known. The potential role of this alternative pathway of processing in the post-translational regulation of viral gene expression is discussed.

Cell Membrane↗
PubMed1992

cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.

Amino Acid Sequence↗
PubMed1992

Conservation of RNA-protein interactions among picornaviruses.

Picornavirus genomes encode unique 5' noncoding regions (5' NCRs) which are approximately 600 to 1,300 nucleotides in length, contain multiple upstream AUG codons, and display the ability to form extensive secondary structures. A number of recent reports have shown that picornavirus 5' NCRs are able to facilitate cap-independent internal initiation of translation. This mechanism of translation occurs in the absence of viral gene products, suggesting that the host cell contains the necessary components for the cap-independent internal initiation of translation of picornavirus RNAs as well as cellular mRNAs. In an attempt to identify some of the perhaps novel cellular proteins involved in this newly discovered mechanism of translation, we utilized RNA mobility shifts assays to identify and characterize interactions that occur between the 5'NCR of poliovirus type 1 (PV1) and cellular proteins. In this report, we describe two separate interactions between RNA structures from the 5' NCR of PV1 and proteins present in extracts from HeLa cells as well as other cell types. We describe the interaction between nucleotides 186 to 220 (stem-loop D) and a cellular protein(s) present in HeLa cell extracts. Mutational analysis of this stem-loop structure suggests that maintenance of a base-paired structure in the lower stem is necessary to present the sequences which directly interact with the protein(s). We also describe the interaction between nucleotides 220 to 460 (stem-loop E) and a cellular protein present in HeLa cell extracts. This RNA binding activity fractionates to a specific ammonium sulfate fraction (A cut) of a ribosomal salt wash. Mutational analysis of the stem-loop E structure suggests that the preservation of an extensive RNA structure is necessary for a strong interaction with the cellular protein(s), although smaller RNAs derived from this region of the 5' NCR can interact to lesser extents. Finally, we show that both of these RNA-protein interactions are conserved among the closely related enteroviruses PV1 and coxsackievirus type B3, human rhinovirus type 14, and the more distantly related cardiovirus Theiler's murine encephalomyelitis virus, suggesting that such RNA-protein interactions serve basic functions which are conserved and utilized by each of these picornaviruses.

Base Sequence↗
PubMed1992

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.

Base Sequence↗
PubMed1991

Poliovirus thiol proteinase 3C can utilize a serine nucleophile within the putative catalytic triad.

The picornavirus 3C proteinases are substrate-specific thiol proteases that have been shown by secondary structure predictions and protein modeling studies to be similar to the trypsin-like serine proteases. We have examined several mutations of the 3C proteinase at putative active site and non-active site residues. The effect on 3C-mediated protein processing supports the model of serine protease similarity. In particular, we have shown that 3C can utilize a serine at position 147, which is predicted to supply the nucleophilic residue of the catalytic triad. We suggest that picornavirus 3C proteinases may represent a class of enzymes that have maintained the catalytic mechanism characteristic of a proposed enzyme ancestral to the highly divergent class of serine proteases.

Cysteine Endopeptidases↗
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↗