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R W Moyer

Publications and source records attributed to R W Moyer.

At least 73 records · Page 4Linked to original sources
PubMed1991

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of two nonessential regions of the rabbitpox virus genome involved in virulence.

We have developed a means to identify genes associated with particular aspects of virulence. By beginning with an avirulent deletion mutant of rabbitpox virus and systematically reintroducing overlapping segments of the deleted region, we have identified two regions of the viral genome associated with increased virulence in mice. Evaluation of illness has been aided by the exploitation of weight loss as an indicator of pathogenesis. One of the regions identified by this method contains several open reading frames and includes two previously described genes. A third, as yet undescribed, gene within this region potentially encodes a product related to the C5 protein of human complement. The second region of DNA associated with increased virulence is the HindIII M fragment, which contains only one complete open reading frame. Analysis of this previously unreported gene shows coding potential for a polypeptide of 254 amino acids (approximately 25 kDa) which is related to the C4 component of human complement. The elucidation of two new viral genes related to complement components, taken together with the recent report of the biological activity of a poxvirus-encoded complement-binding protein, suggests the importance of interactions of the virus with the complement system during a normal infection.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of the vaccinia virus hemagglutinin gene.

The vaccinia virus hemagglutinin (HA) is a glycoprotein found on the plasma membrane of infected cells and the envelope of extracellular virus. Two forms of HA (85 and 68 kDa) are detected by immunoblot analysis. Although hemagglutination activity is only readily detectable late in infection, the 85-kDa HA appears early and accumulates throughout infection, whereas the 68-kDa form appears only late in the cycle. Production of the 68-kDa HA but not the 85-kDa HA was inhibited by either cytosine arabinoside or rifampin. Analysis of HA gene expression reveals a complex pattern of expression. The HA gene is transcribed early to yield a 1.65-kb dicistronic early transcript, consisting of the 945-bp HA open reading frame (ORF) fused to a 453-bp downstream ORF. Transcription from this site initiates 7 bases upstream of the AUG initiating codon of the HA ORF. Due to the discrepancy between the calculated size of the HA protein (33 kDa) and that reported for the unglycosylated HA protein derived from in vitro translation (58 kDa), we placed an early transcription termination signal (TTTTTAT) directly downstream of the 945-bp HA ORF. This led to a reduction in size of the early HA mRNA to 1.2 kb, as expected, but had no effect on the formation of either the 85- or 68-kDa protein. Transcripts originating from the early promoter are found throughout the infection cycle. However, after DNA replication, transcription from a second, late promoter ensues. The transcriptional start site of the late promoter is within a consensus TAAATG sequence located 135 bases upstream of the transcriptional start site of the first promoter. The late transcriptional start site is also found within an upstream ORF.

Amino Acid Sequence↗
PubMed1990

Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression.

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.

Animals↗
PubMed1989

Recruitment to the cytoplasm of a cellular lamin-like protein from the nucleus during a poxvirus infection.

Monoclonal antibodies (Mabs) directed against core proteins of rabbit poxvirus (RPV) have proven effective in the identification of host cell proteins such as RNA polymerase II (Pol II) that may play a role in the infectious process (D. K. Morrison and R. W. Moyer, 1986, Cell 44, 587-596). In this article we describe a Mab that has allowed the detection and characterization of a lamin-like protein derived from the nucleus of the infected cell, which like Pol II is recruited to the cytoplasm following RPV infection. A portion of the gene encoding this protein has been isolated through the screening of a lambda gt11 expression vector library. Sequence analysis of the gene shows it to be derived from a member of the HindIII 1.9-kb repetitive element, a family of mammalian repetitive sequences that are highly conserved. Immunoblot analysis and sequence analysis of the open reading frame show divergent relatedness to certain nuclear lamins. The protein is not, however, one of the three principal lamins characterized to date, but instead appears to be a perinuclear protein related to the highly conserved nuclear lamins that is recruited to the cytoplasm during the infectious process.

Amino Acid Sequence↗
PubMed1989

Interference with vaccinia virus growth caused by insertion of the coding sequence for poliovirus protease 2A.

Attempts were made to express noninfectious derivatives of full-length type 1 (Mahoney) and type 2 (Lansing) poliovirus cDNAs in live recombinant vaccinia viruses for vaccine purposes. Vaccinia virus (VV) would not tolerate insertions of polio cDNA containing the coding sequence for the polio protease 2A. However, polio cDNA with the 2A gene deleted either in vivo or in vitro could be inserted into VV and stably maintained. Genetic evidence indicated that expression of the polio 2A gene in trans from transfected plasmid DNA was deleterious to vaccinia virus within the same cell. The 2A product presumably interferes with VV growth by modifying the host translational machinery such that translation of host and vaccinia capped mRNAs is inhibited. Polio cDNA containing a mutated 2A gene whose product is no longer active in host protein shutoff could be inserted into VV. However, inserts containing the intact mutated 2A gene did not synthesize detectable poliovirus protein, although they did produce polio-specific RNA. Expression of polio-specific protein was detected from a VV-polio recombinant containing cDNA encoding the capsid proteins plus an incomplete 2A gene. These results have implications regarding possible vaccine construction, and suggest a mechanism for interference between polio and vaccinia viruses in mixed infection.

Animals↗
PubMed1988

Poxvirus deletion mutants: virulence and immunogenicity.

Post-vaccinial encephalitis and disseminated vaccinia are major concerns with the use of vaccinia virus recombinants as immunization vectors in man. To identify and characterize possible attenuated poxvirus vectors, rabbitpox virus (RPV) (closely related to vaccinia) and four deletion mutants of RPV were studied for organ tropism, neurovirulence, and protection from wild-type challenge in BALB/c mice. Intraperitoneal (IP) inoculation with 10(7) PFU wild-type (wt) RPV or with two mutants 8 sm and 28 (containing approximately 12 kilobase deletions) showed titers of greater than 10(3) PFU/g tissue in multiple organs. In contrast, IP inoculation of 10(7) PFU of mutants 31 or 23 (containing approximately 30 kilobase deletions) showed markedly reduced growth in all organs. Neurovirulence of wt and mutant RPV was determined by intracerebral (IC) inoculation of mice. Wt and mutants 8 sm, and 28 RPV had LD50 less than 10(2) PFU; in contrast, 31 and 23 had LD50 greater than 10(5) PFU. Finally, 10(6) PFU of mutants 31 or 23, were administered to mice by scarification, the normal route of vaccinia immunization. Both 31 and 23 grew locally in the skin and protected mice challenged IC at 21 days with 100 LD50 of wt RPV, while all unimmunized controls died. We conclude that deletion mutants 31 and 23 demonstrate markedly reduced invasiveness and neurovirulence while retaining immunogenicity. Similar deletion mutations in vaccinia may create avirulent, but effective vaccine vectors for man.

Animals↗
PubMed1987

The role of the host cell nucleus in vaccinia virus morphogenesis.

Despite the fact that cells infected with wild type vaccinia virus synthesize viral DNA and assemble progeny virus particles within the cytoplasm, the host cell nucleus is required for a productive infection. Recent evidence suggests that vaccinia virus selectively recruits components from the host cell nucleus into the cytoplasm for use by the developing virus. One of these components is the largest subunit of the cellular RNA polymerase II (Pol II).

Animals↗
PubMed1986

Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions.

A large (approximately 200 kd) subunit of cellular RNA polymerase II (Pol II) and a virus-encoded subunit of rabbit poxvirus (RPV) RNA polymerase (137 kd) react with common monoclonal antibodies. Hybridization studies with viral and cellular DNA clones confirm that the viral and cellular proteins are related. Following RPV infection, the Pol II subunit is translocated from the nucleus to the cytoplasmic virosomes, then packaged into mature virus, and is found associated with the viral RNA polymerase purified from virions. The results suggest that the cellular Pol II subunit may be directly involved in the transcription of viral genes.

Antibodies, Monoclonal↗
PubMed1985

Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses.

Animals↗
PubMed1985

Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase.

A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment.

Antibodies, Monoclonal↗
PubMed1983

A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis.

A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease. This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

Animals↗
PubMed1983

The white pock mutants of rabbit poxvirus: V. In vitro translation of early host range mutant mRNA.

The abortive infections of pig kidney (PK) cells by both RP mu hr23 and RP mu hr31, two early (DNA minus) white pock (mu) host range (hr) mutants of rabbit poxvirus (RPV), are characterized by the in vivo inhibition of both host and viral protein synthesis by 10 hr postinfection although viral RNA synthesis continues. Further analysis reveals that large quantities of functional viral mRNA can be isolated from PK cells abortively infected with RP mu hr23 and translated in vitro throughout the 10-hr period of infection, even though these mRNAs are almost totally inactive in vivo. The in vitro translation of accumulated mRNA isolated from PK cells abortively infected by RP mu hr31 shows a quite different pattern where the maximum amount of RNA translatable in vitro is found at 6 hr postinfection with lesser amounts at 10 hr postinfection. Although early or prereplicative viral proteins are detected with each mutant both in vivo and after in vitro translation of isolated RNA, few, if any, late proteins are observed under any conditions.

Animals↗
PubMed1981

The mechanism of cytoplasmic orthopoxvirus DNA replication.

Orthopoxvirus DNA replication occurs in the cytoplasm of infected cells within discrete foci designated as virosomes. We show that newly synthesized rabbit poxvirus (RPV) virosomal DNA consists predominantly of concatamers wherein unit length molecules are joined by fusion of two left (LL) or right (RR) ends, resulting in genomes aligned in alternating head-to-head and tail-to tail mirror image arrays. These concatameric molecules serve as the substrates from which unit length DNA molecules are excised during morphogenesis. We propose a mechanism by which internal deletions within these concatameric arrays prior to genome excision and packaging could create inverted terminal repeats and generate gene duplications.

Cytoplasm↗
PubMed1980

Isolation and characterization of a putative bacteriophage T5 transcription.replication enzyme complex from infected Escherichia coli.

A well defined enzyme comples of approximately 5 X 10(6) daltons that contains phage and host cell components known to be required for the processes of phage transcription and DNA replication has been isolated from bacteriophage T5-infected Escherichia coli cells. In addition to the RNA polymerase of the host cell, the complex contains the phage-encoded: gpC2 which has been implicated genetically as a controlling element of late transcription; gpD9, the DNA polymerase required for T5 DNA replication; the proteins gpD5 (DNA-binding protein), and gpD15 (nuclease) which are both known to be essential for T5 DNA replication and for the initiation of late transcription. The viral gpD5 derived from the purified complex is a phosphoprotein. The enzyme complex also contains, protected from the action of nuclease, double-stranded DNA with an approximate molecular weight of 1 to 2 X 10(6) (2 to 3% of the size of the T5 genome) which is derived preferentially from the center of the T5 DNA molecule. The composition of the enzyme complex suggests that the processes of transcription and replication are integrated in T5-infected cells.

DNA Helicases↗