Proteolytic Cleavage of Bovine Adenovirus 3-Encoded pVIII.

Proteolytic maturation involving cleavage of one nonstructural and six structural precursor proteins including pVIII by adenovirus protease is an important aspect of the adenovirus life cycle. The pVIII encoded by bovine adenovirus 3 (BAdV-3) is a protein of 216 amino acids and contains two potential protease cleavage sites. Here, we report that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus protease cleavage sites. Usage of at least one cleavage site appears essential for the production of progeny BAdV-3 virions as glycine-to-alanine mutation of both protease cleavage sites appears lethal for the production of progeny virions. However, mutation of a single protease cleavage site of BAdV-3 pVIII significantly affects the efficient production of infectious progeny virions. Further analysis revealed no significant defect in endosome escape, genome replication, capsid formation, and virus assembly. Interestingly, cleavage of pVIII at both potential cleavage sites appears essential for the production of stable BAdV-3 virions as BAdV-3 expressing pVIII containing a glycine-to-alanine mutation of either of the potential cleavage sites is thermolabile, and this mutation leads to the production of noninfectious virions.IMPORTANCE Here, we demonstrated that the BAdV-3 adenovirus protease cleaves BAdV-3 pVIII at both potential protease cleavage sites. Although cleavage of pVIII at one of the two adenoviral protease cleavage sites is required for the production of progeny virions, the mutation of a single cleavage site of pVIII affects the efficient production of infectious progeny virions. Further analysis indicated that the mutation of a single protease cleavage site (glycine to alanine) of pVIII produces thermolabile virions, which leads to the production of noninfectious virions with disrupted capsids. We thus provide evidence about the requirement of proteolytic cleavage of pVIII for production of infectious progeny virions. We feel that our study has significantly advanced the understanding of the requirement of adenovirus protease cleavage of pVIII.

Adenoviral gene transfer of nitric oxide synthase increases cerebral blood flow in rats.

OBJECTIVE: Depletion of nitric oxide may play a role in the development of vasospasm after aneurysmal subarachnoid hemorrhage. Replenishment of nitric oxide might be a useful treatment for vasospasm. Using rats, we performed intracisternal injections of replication-defective adenovirus containing the endothelial nitric oxide synthase (eNOS) gene and determined the localization of and effect on cerebral blood flow of transgene expression. METHODS: Rats underwent baseline measurement of cortical cerebral blood flow using laser Doppler flowmetry. Replication-defective adenovirus containing the Escherichia coli LacZ gene (Ad327beta-Gal, n = 2/time point) or the bovine eNOS gene (AdCD8-NOS, n = 4/time point) or physiological saline solution was injected into the cisterna magna. Cerebral blood flow was measured 1, 2, 4, 7, or 14 days later, and the animals were killed. Expression of beta-galactosidase activity from the LacZ gene was examined by histochemical staining and that of eNOS was examined by polymerase chain reaction assays of messenger ribonucleic acid. Brains were histopathologically examined for inflammation. RESULTS: Beta-galactosidase activity was observed throughout the leptomeninges and in some cells in the adventitia of small subarachnoid blood vessels in the Ad327beta-Gal-injected rats. Messenger ribonucleic acid for eNOS was detected in the leptomeninges and brainstem 1 and 2 days after injection of AdCD8-NOS. Rats injected with Ad327beta-Gal or physiological saline solution exhibited decreased cerebral blood flow beginning 2 days after virus injection and lasting up to 14 days after injection. Rats injected with AdCD8-NOS developed significant transient increases in cerebral blood flow 2 days after virus injection, followed by slight decreases in blood flow. There was inflammation in the subarachnoid space of all animals; the inflammation was qualitatively worse in animals injected with Ad327beta-Gal, compared with rats injected with AdCD8-NOS or saline solution. CONCLUSION: Intracisternal injection of replication-defective adenovirus containing the eNOS gene can transiently increase cerebral blood flow.

DNA sequencing and phylogenetic analysis of the protease gene of ovine adenovirus 3 suggest that adenoviruses of sheep belong to two different genera.

Until now, the only published ovine adenovirus DNA sequence was the complete genome of ovine adenovirus isolate 287 (OAV287) which, compared to other mammalian adenoviruses, possesses strikingly unique genomic organisation and should properly be classified into a new adenovirus genus. The protease gene sequence of ovine adenovirus type 3 (OAdV-3) was determined and analysed. The results of phylogenetic analysis of the 205 residue long protein demonstrated that OAdV-3 belongs to the genus Mastadenovirus, and is surprisingly closely related to bovine adenovirus type 2. In spite of the common host origin, the evolutionary distance between OAdV-3 and OAV287 proved to be great suggesting that sheep, similarly to cattle and fowl, might be infected by distantly related adenoviruses belonging to different genera.

Sequencing analysis of the region encoding the DNA polymerase of bovine adenovirus serotypes 2 and 3.

Bovine adenoviruses (BAVs) are important pathogens causing significant economic losses to the cattle industry. We have been interested in the differences among serotypes of these viruses, particularly in their pathogenicity and host range. As part of our efforts to better understand these viruses, we have determined the nucleotide sequences for serotype 3 (BAV3) at map coordinates beween 11.7 and 23.7% and for serotype 2 (BAV2) between 13.1 and 24.0%. Analyses of these sequences revealed large open reading frames (ORFs) encoded within the leftward-reading strand of the viral DNA. The coding capacity of the ORF in BAV3 is 1,167 amino acid residues and 1,138 in BAV2. A search in the GenEMBL protein sequence databank for homology to the predicted polypeptide products of these ORFs established their identity as that for the adenovirus (Ad) DNA polymerase (DNA pol). The deduced polypeptide sequences were aligned with each other and with other known Ad DNA pols to reveal regions of homology and similarity. The comparison at the amino acid sequence level not only showed that the bovine Ad DNA pols from the two serotypes are quite distinct from each other, but also revealed that Ad DNA pols contain multiple domains that are highly conserved among human, canine and bovine Ads. These conserved domains are likely important for the multiple functions attributed to Ad DNA pol, which include catalysis of its own initiation complex, elongation of nascent DNA strand, as well as correction of DNA replication errors.

Sequencing and phylogenetic analysis of the protease gene, and genetic mapping of bovine adenovirus type 10 define its relatedness to other bovine adenoviruses.

The complete genome of a bovine adenovirus (BAV) type 10 isolate was molecularly cloned and partially sequenced. The encoded proteins were predicted by computer analysis of the DNA sequences of the ends or the entire length of the cloned viral fragments, and thus a rough genetic map was constructed. The protease gene of BAV-10 was completely sequenced and used in phylogenetic analysis. Based on the results of the phylogenetic analysis, and the location and presence of certain genes thought to be specifically characteristic of subgroup 1 or subgroup 2 BAVs, it could be concluded that, in spite of the striking similarity in certain biological properties, BAV-10 is not related to subgroup 2 BAVs as originally described. It does not however fit clearly into subgroup 1 either, the members of which show closer relationship with human adenoviruses. BAV-10 therefore should best be considered as the first member of a third subgroup of BAVs.

In vivo adenovirus-mediated gene transfer to newborn rat retinal pigment epithelial cells.

A successful surgical access to the subretinal space is critical for achieving adenovirus-mediated gene transfer to the retinal pigment epithelial (RPE) cells or photoreceptor cells. We report a novel surgical approach allowing an efficient delivery of recombinant replication-deficient adenoviral vectors into the subretinal space of newborn rats. Our data suggest that this method may be useful for infecting reproducibly large area of the RPE cell layer of normal newborn rats and should be applicable to RCS pups. We also show the feasibility of infecting ex vivo RPE cells in culture using the same recombinant adenoviral vector.

Nucleotide and amino acid sequence analysis of the porcine adenovirus 23K protein.

The genomic location of the viral encoded protease (23K) of porcine adenovirus serotype 3 (PAV3) was determined and the appropriate fragment cloned and sequenced. An open reading frame (ORF) coding for a polypeptide of 203 amino acids and a calculated molecular weight of 23.3 kDa was found. The ORF was situated in a position similar to that of the human adenovirus 23K, that is, between a putative stop codon for the hexon gene and the polyadenylation signal, AATAAA, for the late region 3. Amino acid sequence alignment of the predicted polypeptide with the sequences of the 23K proteins from other mammalian adenoviruses revealed homology of between 50% and 60% for all except the bovine adenovirus type 7, which displayed appreciable variance from the PAV3 putative 23K with an overall sequence homology of approximately 35%. Conserved cysteine, histidine and proline residues believed to be important in the activity of the 23K protein of human adenoviruses were also present in the PAV3 protein. The genomic location and amino acid sequence of the characterised reading frame suggests that this gene is that of the 23K protein of PAV3.

The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis.

Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95%. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.