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M R Denison

Publications and source records attributed to M R Denison.

18 recordsLinked to original sources
PubMed1998

Processing of the MHV-A59 gene 1 polyprotein by the 3C-like proteinase.

The 3C-like proteinase of mouse hepatitis virus (MHV-3CLpro) is predicted to cleave at least 10 sites in the gene 1 polyprotein, resulting in processing of proteinase, polymerase and helicase proteins from the polyprotein. We have used E. coli expressed recombinant 3CLpro (r3CLpro) to define cleavage sites in carboxy-terminal region of the ORF 1a polyprotein. Polypeptides containing one or more putative 3CLpro cleavage site were translated in vitro from subcloned regions of gene 1, and the polypeptides were incubated with r3CLpro. Analysis of the cleavage products confirmed several putative cleavage sites, as well as identifying cleavage sites not previously predicted by analysis of the MHV sequence. Antibodies directed against a portion of the ORF 1a polyprotein were used to probe virus infected cells, and detected proteins that correspond to the cleavage sites used by 3CLpro in vitro. These results suggest that MHV 3CLpro cleaves at least 7 sites in the ORF 1a polyprotein, and that the specificity of 3CLpro for cleavage site dipeptides may be broader than previously predicted.

Animals↗
PubMed1998

Expression, purification, and activity of recombinant MHV-A59 3CLpro.

The 3C-like proteinase (3CLpro) of MHV-A59 is predicted to mediate the majority of proteolytic processing events within the gene 1 polyprotein. We have overexpressed 3CLpro in E. coli as a fusion protein with maltose binding protein (MBP). The MBP-3CLpro fusion protein was purified from contaminating E. coli proteins by amylose column chromatography, and r3CLpro was cleaved from the fusion protein by factor Xa. Recombinant 3CLpro (r3CLpro) was able to cleave a polypeptide substrate containing mutated inactive 3CLpro and portions of the flanking domains. R3CLpro cleaved substrate completely within 5 minutes and the activity of r3CLpro was sensitive to inhibition by serine and cysteine proteinase inhibitors; however, it was not inhibited by EDTA, suggesting that metal ions were not critical for 3CLpro activity.

Animals↗
PubMed1998

Expression and processing of nonstructural proteins of the human astroviruses.

The human astroviruses (HAst) are increasingly recognized as an important cause of gastroenteritis. These viruses contain a 6.8-kb positive-sense, single-stranded RNA molecule that is infectious when transfected into permissive cells. The HAst gene 1 is composed of two open reading frames (ORFs 1a and 1b) connected by a ribosomal frameshift. Gene 1 is predicted to encode two nonstructural polyproteins (pp 1a and pp 1ab), and analysis of the HAst gene 1 sequence has resulted in predictions of a serine proteinase within the ORF1a polyprotein. However, none of the gene 1 proteins have been identified. To examine the expression and processing of the HAst2 gene 1 polyprotein, we have translated pp 1a and pp 1ab in vitro. These ongoing studies will provide the foundation for correlating gene 1 expression in vitro with proteins expressed in virus-infected cells.

Antibodies, Viral↗
PubMed1998

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

3C Viral Proteases↗
PubMed1997

Determinants of mouse hepatitis virus 3C-like proteinase activity.

The coronavirus, mouse hepatitis virus strain A59 (MHV), expresses a chymotrypsin-like cysteine proteinase (3CLpro) within the gene 1 polyprotein. The MHV 3CLpro is similar to the picornavirus 3C proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of Q/(S, A, G) dipeptide cleavage sites. However, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus 3C-like proteinases or of the precise coding sequence of 3CLpro within the gene 1 polyprotein. In this study, aspartic acid residues in MHV 3CLpro were mutated and the mutant proteinases were tested for activity in an in vitro trans cleavage assay. MHV 3CLpro was not inactivated by substitutions at Asp3386 (D53) or Asp3398 (D65), demonstrating that they were not catalytic residues. MHV 3CLpro was able to cleave at a glutamine-glycine (QG3607-8) dipeptide within the 3CLpro domain upstream from the predicted carboxy-terminal QS3636-6 cleavage site of 3CLpro. The predicted full-length 3CLpro (S3334 to Q3635) had an apparent mass of 27 kDa, identical to the p27 3CLpro in cells, whereas the truncated proteinase (S3334 to Q3607) had an apparent mass of 24 kDa. This 28-amino-acid carboxy-terminal truncation of 3CLpro rendered it inactive in a trans cleavage assay. Thus, MHV 3CLpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3CLpro domain was required for activity. These studies suggest that the coronavirus 3CL-proteinases may have a substantially different structure and catalytic mechanism that other 3C-like proteinases.

3C Viral Proteases↗
PubMed1996

Intracellular and in vitro-translated 27-kDa proteins contain the 3C-like proteinase activity of the coronavirus MHV-A59.

The coronavirus mouse hepatitis virus-A59 (MHV-A59) encodes a serine-like proteinase (3C-like proteinase or 3CLpro) in ORF 1a of gene 1 between nucleotides 10,209 and 11,114. We previously have demonstrated that proteins expressed in vitro from a cDNA clone of the 3CLpro region possess proteinase activity, and that the proteinase is able to cleave substrate in trans. We sought to determine if the 27-kDa in vitro cleavage product (p27) was an active form of the 3CLpro and whether this was consistent with the 3CLpro expressed in virus-infected cells. Antibodies directed against the 3CLpro domain detected 27-kDa MHV proteins in vitro and in MHV-A59-infected cells. The 27-kDa proteins were able to cleave substrate in trans without other protein cofactors or supplemental membranes, and the p27 proteinase activity was retained after purification by immunoprecipitation and gel electrophoresis. When p27 was expressed in vitro with portions of the amino-and carboxy-terminal flanking domains (MP1 and MP2), p27 was not liberated by cls cleavage. The proteolytic activity of the 27-kDa proteins was inhibited by a variety of cysteine and serine proteinase inhibitors, and was eliminated by the cysteine proteinase inhibitor E64d. These results indicate that the 27-kDa protein is a mature proteinase in MHV-A59-infected cells, and that appropriate processing of this molecule occurs in vitro.

Amino Acid Sequence↗
PubMed1996

Veillonella parvula bacteremia without an underlying source.

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal human flora. It has rarely been identified as a pathogen in humans, and the most frequently reported infection caused by V. parvula is osteomyelitis. We report a case of bacteremia unrelated to a central venous catheter and without an underlying source of infection.

Bacteremia↗
PubMed1995

Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine coronavirus MHV-A59.

A 65-kDa protein has been detected in mouse hepatitis virus A59 (MHV-A59)-infected DBT cells using polyclonal antibodies directed against polypeptides encoded by the 5' 1.8 kb of gene 1. The presence of this 65-kDa protein (p65) was previously predicted from immunoprecipitation studies of gene 1 expression in MHV-A59-infected DBT cells with other antisera (1). p65 was rapidly labeled in virus-infected cells at late times of infection; however, its cleavage from the polyprotein was significantly delayed compared to the amino-terminal gene 1 polyprotein cleavage product, p28. Similar to p28, p65 was cleaved from the growing polyprotein without detectable intermediate precursors. Kinetic analysis of p65 with specific antibodies indicates that p65 is immediately adjacent to p28 in the gene 1 polyprotein. The proteolytic activity responsible for the carboxy-terminal cleavage of p65, as well as the function of the p65 protein, remains to be determined.

Animals↗
PubMed1995

Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59.

Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, and E. V. Koonin, FEBS Lett. 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res. 17:4847-4861, 1989). We have cloned into a T7 transcription vector a cDNA fragment containing the putative 3C-like proteinase domain of MHV-A59, along with portions of the flanking hydrophobic domains. The construct was used to express a polypeptide in a combined in vitro transcription-translation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecular masses of 32 and 27 kDa were predominant after 30 to 45 min and appeared to be products of specific proteolysis of larger precursors. Mutations at the putative catalytic histidine and cysteine residues abolished the processing of the 27-kDa protein. Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine mutants. The amino-terminal cleavage of the 27-kDa protein occurred at a glutamine-serine dipeptide as previously predicted. This study provides experimental confirmation that the coronaviruses express an active proteinase within the 3C-like proteinase domain of gene 1 ORF 1a and that this proteinase utilizes at least one canonical QS dipeptide as a cleavage site in vitro.

Amino Acid Sequence↗
PubMed1994

Coronavirus polyprotein processing.

MHV gene 1 contains two ORFs in different reading frames. Translation proceeds through ORF 1a into ORF 1b via a translational frame-shift. ORF 1a potentially encodes three protease activities, two papain-like activities and one poliovirus 3C-like activity. Of the three predicted activities, only the more amino terminal papain-like domain has been demonstrated to have protease activity. ORF 1a polypeptides have been detected in infected cells by the use of antibodies. The order of polypeptides encoded from the 5' end of the ORF is p28, p65, p290. p290 is processed into p240 and p50. Processing of ORF1a polypeptides differs during cell free translation of genome RNA and in infected cells, suggesting that different proteases may be active under different conditions. Two RNA negative mutants of MHV-A59 express greatly reduced amounts of p28 and p65 at the non-permissive temperature. These mutants may have defects in one or more viral protease activities. ORF 1b, highly conserved between MHV and IBV, potentially contains polymerase, helicase and zinc finger domains. None of these activities have yet been demonstrated. ORF 1b polypeptides have yet been detected in infected cells.

Animals↗
PubMed1993

A newly identified MHV-A59 ORF1a polypeptide p65 is temperature sensitive in two RNA negative mutants.

Polypeptide products of MHV-A59 gene 1 have been identified in infected DBT cells and in the products of in vitro translations of genome RNA. In this paper we report the identification in infected cell lysates of a 65-kDa polypeptide (p65) encoded in ORF 1a. Studies on the kinetics of appearance and processing of p65 show that p65 is detectable after p28 but before the appearance of p290, p240 and p50. No homologue of the p65 polypeptide identified in infected cell lysates was immunoprecipitated from in vitro translations of genomic RNA, providing further evidence that in vitro processing of polypeptides encoded in ORF 1a of gene 1 differs from that which occurs late in infection of DBT cells. Although the function of p65 is unknown, two MHV-A59 ts mutants isolated and characterized by Baric et al. (3,4) do not produce detectable levels of p65 at the non-permissive temperature indicating that p65 may play an important role in the virus life cycle.

Animals↗
PubMed1992

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events.

Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

Leupeptins↗
PubMed1991

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.

Amino Acid Sequence↗
PubMed1986

Translation and processing of mouse hepatitis virus virion RNA in a cell-free system.

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.

Animals↗
PubMedDate not supplied

Fatal fat embolism syndrome in a child with undiagnosed hemoglobin S/beta+ thalassemia: a complication of acute parvovirus B19 infection.

Anemia, mental status changes, and fatal respiratory failure complicated a febrile illness in a previously healthy 14-year-old black female. At autopsy, widespread fat emboli and bone marrow necrosis were found. Hemoglobin electrophoresis on an antemortem, pretransfusion specimen revealed hemoglobin S/beta+ thalassemia. Acute parvovirus B19 (PV B19) infection was suspected. Postmortem serum and a variety of paraffin-embedded tissues were assayed for PV B19 DNA using the polymerase chain reaction (PCR). The expected PCR product was identified in the serum specimen and in paraffin-embedded sections of bone marrow, kidney, spleen, parathyroid, thyroid, adrenal, and gastrointestinal tract: lung, liver, ovary, fallopian tube, uterus, brain, heart, and pancreas were negative. PV B19 infection is highly contagious and may be rapidly fatal in children with hemoglobinopathies by several mechanisms, including fat embolism. Therefore, there exists the risk of multiple deaths within a family. The acute infection may be easily and expeditiously diagnosed using serum or a variety of paraffin-embedded tissues.

Acute Disease↗