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B L Semler

Publications and source records attributed to B L Semler.

86 records · Page 5Linked to original sources
PubMed1982

A membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide.

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNa polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNa replication complex.

Antibodies, Viral↗
PubMed1982

Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs.

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7c. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH2-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. We conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c (or P3-2 and P3-6a) and not in P2-X.

Amino Acid Sequence↗
PubMed1982

Cleavage sites within the poliovirus capsid protein precursors.

Partial amino-terminal sequence analysis was performed on radiolabeled polio-virus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.

Amino Acid Sequence↗
PubMed1981

Primary structure, gene organization and polypeptide expression of poliovirus RNA.

The primary structure of the poliovirus genome has been determined. The RNA molecule is 7,433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2,207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

Amino Acid Sequence↗
PubMed1981

Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing.

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region (P3) of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occurs between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.

Amino Acid Sequence↗
PubMed1981

Stable hairpin structure within the 5'-terminal 85 nucleotides of poliovirus RNA.

The primary sequence of a 5'-terminal fragment of poliovirus type 1 RNA, generated by digestion with RNase III, has been determined. This sequence reveals the presence of a stable hairpin structure beginning nine nucleotides from the terminally linked protein VPg. The sequence does not contain (i) the initiation codons AUG or GUG or (ii) the putative ribosome-binding sequence complementary to the 3' end of eucaryotic ribosomal 18S RNA. The stem-and-loop structure identified can be drawn in either plus or minus RNA strands. It is unclear to which strand functional significance (if any) can be assigned. It is possible that the hairpin structure is involved in ribosomal recognition and translation or in RNA synthesis by interacting with replicase molecules.

Base Sequence↗
PubMed1980

Protein-linked RNA of poliovirus is competent to form an initiation complex of translation in vitro.

Poliovirus RNA that had been labelled with 125I in the 5'-terminal protein (VPg) was found competent to form an initiation complex of translation in a cell-free reticulocyte lysate. In conditions of ribosome binding, no cleavage occurred between VPg and RNA. We conclude that removal of VPg from poliovirus RNA is not a prerequisite for this RNA to initiate translation in vitro.

Cell-Free System↗
PubMed1979

The nucleotide sequence of the 5' terminus of vesicular stomatitis virus RNA.

We have determined the nucleotide sequence for the first 50 nucleotides at the 5' terminus of vesicular stomatitis virus (VSV) genome RNA. This sequence is identical to that of the in vitro RNA polymerase product synthesized by defective interfering (DI) particles of VSV. These results confirm previous conclusions rengarding DI and standard viral terminal sequences based on hybridization studies and earlier sequencing of the DI polymerase product RNA.

Animals↗
PubMed1979

Evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus.

Persistent infection of BHK21 cells was established with cloned vesicular somatitis virus plus purified Dl particles and maintained in vitro for over 5 years. After 1 year of persistence, the infectious virus RNA genome had evolved several oligonucleotide map changes, and numerous changes had accumulated by 3.5 years. Additional evolution occurred by the fourth year and continued until the fifth year. In contrast, repeated passage of virus in acute infections of several cell types in vitro or in vivo did not lead to detectable oligonucleotide map changes. The short Dl particle originally used to co-infect with infectious virus in establishing persistent infection has been displaced by an ever present and constantly changing population of other Dl particles of differing sizes and radically differing oligonucleotide maps. We conclude that the genomes of both infectious VSV and its Dl particles undergo continuous evolutionary change during years of persistence. In the infectious virus, these changes involve hundreds of mutations which are usually expressed as poorly replicating, temperature-sensitive, small plaque mutants. These are stable mutants which do not revert to wild-type when passaged repeatedly in acute infections at 37 or 33 degrees C. It appears that the sequestered intracellular environment of persistently infected cells favors rapid and continuous virus evolution.

Animals↗
PubMed1979

Internal genome deletions in two distinct classes of defective interfering particles of vesicular stomatitis virus.

We have characterized the genome sequences represented in two defective interfering particles derived from the heat-resistant strain of vesicular stomatitis virus by means of end-labeling and hybridization techniques. Both defective particle RNAs, which differ slightly in size, contain 5'-end sequences identical to each other and to that of the standard infectious virus genome, for at least the first approximately 55 bases. In contrast, the 3'-end sequences of these two RNAs are different. The 3'-end sequence of the smaller RNA is identical to that of the standard genome for at least the first 48 bases. The 3'-end sequence of the larger RNA is an inverted complement of its 5' end for approximately 65 bases. The bulk of the sequences in both RNAs is derived from the 3' half of the standard genome. We also show that the two defective particles differ in vitro transcription and in vivo replication properties. These results provide direct evidence for the presence of internal genome deletions in defective interfering particles of negative-stranded RNA animal viruses and demonstrate the existence of at least two distinct classes of these particles.

Chromosome Deletion↗
PubMed1979

Persistent vesicular stomatitis virus infection mediates base substitutions in viral RNA termini.

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.

Animals↗
PubMed1978

Sequence of a RNA templated by the 3'-OH RNA terminus of defective interfering particles of vesicular stomatitis virus.

We have sequenced the endogenous RNA polymerase product produced by disrupted purified virions of vesicular stomatitis virus defective interfering particles by using the newer one-dimensional rapid gel sequencing techniques and confirming this with a modified two-dimensional gel vectoring technique. The sequence of this 46-nucleotide RNA is: 5'(pp)pACGAAGACCACAAAACCA-GAUAAAAAAUAAAAACCACAAGAGGG(U)COH3'. We infer that this sequence is identical to the sequence at the 5' end of infectious vesicular stomatitis virus RNA and is complementary to the sequence of the 3'-OH terminus of this defective interfering particle genome RNA.

Base Sequence↗
PubMed1975

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.

Acetamides↗