Ahasan, Mohammad Mainul
(2008).
Characterisation of open reading frames m29 and m29.1 in murine cytomegalovirus.
University of Birmingham.
Ph.D.
Abstract
Murine cytomegalovirus (MCMV) in its natural host, the mouse, is an excellent model for studying the biology of cytomegalovirus infection. Mostly this model has been used to study gene homologues of human cytomegalovirus (HCMV). Of the predicted 170 MCMV open reading frames (ORFs) only 78 have significant amino acid identity with genes in HCMV. To better understand the biological mechanisms underlying the differences between the viruses, for example their species specificity and immune evasion genes, MCMV unique ORFs need to be examined. Here the role of m29 and m29.1 ORFs in the MCMV (Smith strain), which have no homology with ORFs of any other cytomegalovirus, have been examined. The m29 and m29.1 ORFs are overlapping and encoded on opposite strands of the double-stranded DNA genome. Sequence analysis over this region showed a discrepancy to the published sequence. An additional G (guanine) nucleotide was found at nucleotide position 36,198 that alters the predicted ORFs, m29 being 242 amino acids shorter and m29.1 210 amino acids longer than the predicted sequence. This was confirmed by sequencing the MCMV Birmingham K181 strain, the Birmingham Smith strain and MCMV wild type isolates- N1, K17A and G4. Transcripts from the newly identified m29 and m29.1 ORFs were confirmed by reverse transcriptase PCR (RT-PCR). They were produced at early (3h) and immediate-early (2h) times post-infection respectively as determined by cycloheximide and phosphonoacetic acid treatment but were continuously expressed up to at least 24h post-infection. 5' and 3'-RACE (rapid amplification of cDNA ends) analysis from m29.1 ORF confirmed the production of a ~2.4 kb transcript and a low abundance spliced transcript from which a 123bp intron had been removed. Mutants of ORF m29 and m29.1 have been produced in which ET recombination was used to introduce stop codon mutations within these overlapping ORFs. This was achieved by single base alterations near to the 5` end of each ORF that prevented translation but not transcription of each ORF individually. Linear dsDNAs containing the mutations were introduced into the Smith MCMV BAC replacing an antibiotic cassette that had been inserted into the gene of interest. Mutant viruses, Rc29 and Rc29.1 respectively, were recovered from these mutant BACs by in vitro passage in tissue culture cells. Revertant virus (Rv29.1) was made by a further 2 step process in which the mutant m29.1 ORF was first replaced by the antibiotic cassette and then by the wt ORF. These mutants were characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Both mutants produced their expected transcripts but Rc29.1 virus produced no corresponding protein as examined by western blot using an antibody produced in rabbits to bacterially expressed protein. Failure to express the m29 ORF in bacteria and failure of a synthetic peptide to generate rabbit antibodies that bound to denatured m29 protein meant that protein expression of the m29 gene in either mutant could not be determined. Mutant virus Rc29 replicated similarly to wild type virus both in tissue culture and in BALB/c mice. Mutant virus Rc29.1 replicated poorly with lower yields, a delay of about 2-3 days in reaching peak titres and an earlier decline compared to wt and revertant (Rv29.1) virus in tissue culture. Rc29.1 virus also showed delayed replication in the salivary glands of BALB/c mice compared to wt and Rv29.1 viruses and in SCID mice peak titres occurred later and mice became sick and had to be humanely killed approximately 8 days later that mice infected with wt virus. These results suggest that m29 and m29.1 ORFs are dispensable for viral replication in vitro in NIH 3T3 cells and in animal hosts. However, the m29.1 ORF is required for optimal viral growth in vitro and in vivo.
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