Biomedical subjects

B L Semler

Publications and source records attributed to B L Semler.

At least 73 records · Page 4Linked to original sources

PubMed1988

Construction of a "mutagenesis cartridge" for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants.

By following a strategy of genetic analysis of poliovirus, we have constructed a synthetic "mutagenesis cartridge" spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type 1 (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed us to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. We suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

Cloning, Molecular↗

PubMed1988

Mutational analysis of the genome-linked protein VPg of poliovirus.

Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.

Amino Acid Sequence↗

PubMed1987

In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase.

We describe a completely in vitro system for generating defined poliovirus proteinase mutations and subsequently assaying the phenotypic expression of such mutations. A complete cDNA copy of the entire poliovirus genome has been inserted into a bacteriophage T7 transcription vector. We have introduced proteinase and/or cleavage site mutations into this cDNA. Mutant RNA is transcribed from the altered cDNA template and is subsequently translated in vitro. Employing such a system, we provide direct evidence for the bimolecular cleavage events carried out by the 3C proteinase. We show that specific genetically-altered precursor polypeptides containing authentic Q-G cleavage sites will not act as substrates for 3C either in cis or in trans. We also provide evidence that almost the entire P3 region is required to generate 3C proteinase activity capable of cleaving the P1 precursor to capsid proteins. However, only the 3C portion of P3 is required to generate 3C proteinase activity capable of cleaving P2 and its processing products.

3C Viral Proteases↗

PubMed1987

Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase.

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.

Amino Acid Sequence↗

PubMed1987

Expression of the poliovirus genome from infectious cDNA is dependent upon arrangements of eukaryotic and prokaryotic sequences in recombinant plasmids.

The introduction of a cDNA copy of poliovirus type 1 (Mahoney) into cultured primate cells results in the production of infectious virus. The level of infectious virus can be increased by incorporation of eukaryotic signals of transcription and replication. We have utilized the SV40 DNA sequence coding for the early and late promoters, the SV40 origin of replication, and the enhancer elements, along with the cDNA of poliovirus, to determine the important parameters for the level of infectious virus produced following transfection. Although plasmid replication increases the level of infectivity, the major determinant of infectivity is promoter activity.

Animals↗

PubMed1987

Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins.

We generated defined alterations in poliovirus protein-processing substrates and assayed the effects of these alterations with an in vitro expression system. A complete cDNA copy of the poliovirus genome was inserted into a bacteriophage T7 transcription vector. Using this expression template, we produced RNA transcripts containing defined regions of the poliovirus capsid precursor polypeptide (P1) and RNA transcripts containing mutations in the P1 and P2 regions. In vitro translation of P1-derived transcripts allowed us to characterize the 3C-mediated cleavage of P1 to capsid proteins. We demonstrated that, for either posttranslational or cotranslational cleavage at any of the Q-G amino acid pairs within P1, almost the entire P1 precursor is required. We also demonstrated that minimal sequences 3' to the 2A coding sequence are required to generate active 2A proteinase in vitro and that two specific four-amino-acid insertions in protein 2C do not alter 2A- or 3C-mediated processing of the poliovirus polyprotein. In addition, we demonstrated that substantial deletion of P1 sequences does not alter 2A-mediated cleavage of the Y-G site at the P1-P2 junction. These results allowed us to compare the P1 sequences required for 2A- versus 3C-mediated processing of the capsid precursor, and we discuss these results in the context of the three-dimensional structure of the capsid proteins.

Amino Acid Sequence↗

PubMed1987

Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase.

We used a synthetic double-stranded oligonucleotide to introduce amino acid substitutions into the proteinase 3C region of a poliovirus type 1 cDNA clone. The six different mutant viruses recovered exhibited a small-plaque phenotype when assayed on HeLa cells. Further investigation revealed that all the mutations (with the exception of one) yielded P3 region proteins that displayed altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A conservative Val----Ala change at amino acid 54 of the proteinase resulted in a virus that was deficient in the production of the mature viral RNA polymerase 3D. Although this mutant achieved less than one-half of the wild-type levels of RNA synthesis during the course of infection, it still grew to nearly wild-type titers.

Amino Acid Sequence↗

PubMed1986

An infectious cDNA clone of the poliovirus Sabin strain could be used as a stable repository and inoculum for the oral polio live vaccine.

Viruses were recovered from HeLa S3 cells and African green monkey kidney (AGMK) cells transfected with an infectious cDNA clone of poliovirus vaccine Sabin 1 strain. The viruses recovered from the different DNA-transfected cells were tested for the biological characteristics of temperature sensitivity (rct marker), plaque size, and bicarbonate concentration dependency (d marker). The results revealed that the above properties were similar to those obtained from tests on the Sabin 1 vaccine reference strain. The recovered viruses and the vaccine reference virus were passaged in AGMK cells at an elevated temperature of 37.5 degrees, and the passaged isolates were tested for the rct marker. The virus recovered from AGMK cells had the most stable rct phenotype while the virus from HeLa S3 cells had a similar stability to that of the reference virus, suggesting that the virus from AGMK cells would be more suitable as a vaccine strain than the other two viruses. Furthermore, an infectious cDNA clone of high specific infectivity, constructed by introducing SV40 large T antigen into the plasmid, was used for production of high titers of virus after transfection. The results of in vitro biological tests on the recovered virus suggested that virus produced in the transfected AGMK cells also had the high quality that is desirable in vaccine stocks. Monkey neurovirulence tests performed with these recovered viruses revealed that the recovered viruses were weakly neurovirulent, similar to the vaccine reference virus. The infectious cDNA clone of the poliovirus vaccine strain could therefore be used to generate a possible inoculum of the oral polio live vaccine. Our findings strongly suggest that an infectious cDNA clone of poliovirus RNA may be used to preserve the constancy and quality of the present seed viruses of the Sabin 1 vaccine strain.

Animals↗

PubMed1986

A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus.

Animals↗

PubMed1986

Genetic analysis of the attenuation phenotype of poliovirus type 1.

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.

Animals↗

PubMed1985

Factors affecting the detection of enteroviruses in cerebrospinal fluid with coxsackievirus B3 and poliovirus 1 cDNA probes.

Enteroviruses are common pathogens of meningitis and encephalitis, and infections are often difficult to distinguish clinically from bacterial and herpetic infections of the central nervous system. An array of enteroviruses added to cerebrospinal fluid in reconstruction experiments were detected by a dot hybridization assay. Optimal handling and processing conditions for infected cerebrospinal fluid were established, and the effect on the hybridization reaction of humoral and cellular components of the inflammatory response was determined. Six hybridization probes, derived from poliovirus 1 and coxsackievirus B3, were then tested, singly and in combinations, to optimize the sensitivity and spectrum of the assay. Implications for enteroviral taxonomy based on these experiments are discussed.

DNA↗

PubMed1985

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.

Bicarbonates↗

PubMed1984

Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals.

Sub-genomic cDNA clones representing the entire genomic RNA of poliovirus Type 1 (Mahoney) have been isolated in E. coli. Construction of a complete cDNA copy of the poliovirus genome in the EcoRI site of plasmid vector pBR325 from these clones is described. Introduction of plasmid DNA containing the complete cDNA copy of polio RNA into cultured primate cells by transfection produces infectious poliovirus. The virus produced by such a transfection appears to be identical to wild type poliovirus. Isolation of a polio recombinant plasmid containing SV40 transcription and replication signals is also described. Transfection of COS-1 cells with this plasmid yields greater than 1,600 plaque-forming units (PFU) per microgram of input DNA.

Base Sequence↗

PubMed1984

Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase.

The poliovirus polyprotein is proteolytically processed predominantly by a virus-encoded proteinase (P3-7c) that cleaves glutamine-glycine amino acid pairs. The biosynthesis of the viral proteinase, itself a product of glutamine-glycine cleavages, was studied by constructing a bacterial expression plasmid that contained a cloned segment of the poliovirus genome slightly larger than the coding region for P3-7c. The induction of expression of this plasmid in E. coli produced several poliovirus-specific polypeptides. One polypeptide, an unstable protein called 3i, was the product of fortuitous in-phase initiation of translation within the coding region of P3-7c. Three other induced polypeptides were products of proteolytic cleavages, the smallest (polypeptide 3) having the properties (amino-terminal amino acids, carboxy-terminal amino acids, size, antigenicity) of P3-7c. Insertion of a DNA linker into the P3-7c coding region results in the loss of P3-7c-specific glutamine-glycine cleavage activity. We conclude that P3-7c was produced by autocatalytic cleavage.

Amino Acid Sequence↗

PubMed1984

Protein processing map of poliovirus.

Five previously unmapped proteins (5a, 7d, 8, 9b, and 10) were located on the proteolytic processing map of the polyprotein. One of the proteins, 9b, appears to be the sister fragment of a cleavage reaction (P3-9 leads to P3-9b + VPg). Two of the other newly mapped proteins, 8 and 10, have been identified as sister fragments of X-related proteins 3b and 5b; thus, P2-3b leads to P2-8 + P2-5b and P2-5b leads to P2-10 + P2-X. The remaining proteins, 5a and 7d, mapped in the 1b protein and appear to result from the cleavages P3-1b leads to P3-5a + P3-6b and P3-4b leads to P3-7d + P3-6b. These assignments account for over 95% of the total polioviral proteins and complete the mapping of the major processing pathways.

Amino Acid Sequence↗

PubMed1984

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.

Animals↗

PubMed1983

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides.

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 and P3 regions of the polyprotein. Sequence analysis of stepwise Edman degradation show that these proteins are 3b/9 (Mr 77,000) and X/9 (Mr 50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.

Amino Acid Sequence↗

PubMed1983

Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition.

The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor of the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. We have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glycine amino acid pair not previously reported to be a viral cleavage site.

Antigen-Antibody Complex↗