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David Rowlands collection

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David Rowlands collection
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Each year, the Microbiology Society Council offer Honorary Membership to distinguished microbiologists who have made a significant contribution to the science. In 2019, David J. Rowlands (Emeritus Professor of Virology, University of Leeds) was appointed an Honorary Member.

This collection brings together Journal of General Virology articles authored by David Rowlands.  


Collection Contents

7 results

    • Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

      We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3D). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3D aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3D (detected by immunoblotting) and shown to be dose dependent. The 3D aptamers appeared to be more effective than 2′--methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.

    • Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system

      We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines ( McCormick , 2002 ). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a -expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5′UTR positioned −2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5′ end of the transcript (5′HH) that cleaves between the ribozyme–5′UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the -interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.

    • The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis

      Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit . This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system – under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

    • translation of hepatitis A virus subgenomic RNA transcripts

      A subgenomic cDNA clone from hepatitis A virus strain HM175, composed of the last eight nucleotides of the 5′ non-translated region and the first 2248 nucleotides of the coding sequence (P1 region), was inserted into a vector under the control of the T7 promoter. Restriction enzyme digestion at sites within the structural region and subsequent transcription yielded RNA products which were translated efficiently in rabbit reticulocyte lysates to produce proteins of the predicted sizes. The translation products were specifically precipitated with antipeptide antisera; these reactions were not affected by denaturation of the antigens by boiling in 1% SDS. The translated proteins were also precipitated by antivirion antisera, but recognition was totally abolished following denaturation. Thus antivirion antisera recognized conformation-dependent epitopes expressed on the translated products exclusively.

    • Immunological Priming with Synthetic Peptides of Foot-and-Mouth Disease Virus

      SUMMARY

      A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein VP1 from foot-and-mouth disease virus (FMDV), serotype O, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141–160 or 141–160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141–160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141–160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response.

    • Immunogenic and Cell Attachment Sites of FMDV: Further Evidence for their Location in a Single Capsid Polypeptide

      SUMMARY

      Chymotrypsin cleaves only one of the four major polypeptides of foot-and-mouth disease virus (FMDV serotype O) . This polypeptide (VP1, mol. wt. 29 × 10) was first cleaved into fragments of mol. wt. 20 and 9 × 10 and further cleavage could be prevented by the addition of a large excess of bovine serum albumin. The infectivity of the virus particles at this stage was the same as that of the intact virus although the rate of attachment to BHK 21 cells was slower and the immunogenic activity was reduced. If hydrolysis was allowed to continue, VP1 was cleaved into fragments with mol. wt. r8 and < 9 × 10, similar to those obtained with trypsin, and the virus particles then had a greatly reduced infectivity and a lower immunogenicity. Treatment of strains from five other serotypes of the virus with the two enzymes cleaved only VP1 in each instance and there was a corresponding loss of infectivity. The results are discussed in relation to the location and biological activity of the virus polypeptides.

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