Adenovirus Type 40 Host Range in Tissue Culture: Replication and Gene Expression in INT407 Cells

The enteric adenoviruses show a restricted host range in tissue culture. Adenovirus type 40 (Ad40) can not be passaged in conventional human cell lines used for other adenovirus serotypes, but will grow in cells constitutively expressing Ad2 or Ad5 ElB 55K function (KB 16 & 293 cells respectively). Preliminary experiments showed that Ad40 could also be propagated in INT407 cells which were not known to express El proteins. The aim of the work described here was to characterise the growth of Ad40 in INT407 cells by examining virus replication and gene expression compared to other permissive (KB16) and semi-permissive cell lines (HeLa & A549). A timecourse of DNA replication confirmed that INT407 cells were permissive for Ad40. The pattern of DNA replication was similar on KB 16 and INT407 cells although the yield from KB16 cells was slightly higher than INT407 cells; the lowest yield was from HeLa. In addition INT407 cells could partially complement the growth of an Ad2/5 55K mutant virus. These results suggest that ElB 55K may be complemented or that the requirement for 55K may vary in INT407 cells. The kinetics of Ad40 growth were studied by fluorescent focus assay using a monoclonal antibody against Ad5 hexon. Ad40 exhibited one hit kinetics on permissive cells (INT407 & KB 16) and two hit kinetics on semi-permissive cells (A549 & HeLa). There was a marked difference in the number of productively infected cells between cell types with fewer cells productively infected at 48h p.i. on semi-permissive cell lines. The entry events of the Ad40 replicative cycle (i.e. attachment and internalisation) were investigated to determine if any defects at this stage might contribute to the Ad40 growth phenotype on the different cell lines. A similar number of Ad40 particles attached and were internalised on INT407 and HeLa cells. These results could not explain why INT407 cells were more permissive for Ad40 growth than HeLa cells. In a parallel experiment the attachment and internalisation of dl309 was found to be comparable to Ad40 on these cell lines. In order to explain the Ad40 growth phenotype in the different cell types ElB gene expression was analysed by Northern blotting and SI nuclease digestion on the different cell lines. ElB 22S mRNA was observed at early times before the onset of DNA replication in INT407 and KB 16 cells, with the 14S mRNA predominantly late in infection in these cell types. In HeLa cells ElB mRNA was detected coincident with the onset of DNA replication at much reduced levels. A similar pattern to HeLa was seen on A549 cells. The pattern of ElB transcription on INT407 cells was comparable to that reported previously on KB 16 cells which is similar to Ad 12 with 14S, 22S and 9S mRNA. The lower levels of ElB mRNA and later detection in HeLa cells may indicate that the transcription from the ElB promoter is inefficient or absent early in infection in these cells. Even in permissive cells Ad40 ElB mRNA transcription is poor compared to Ad5 which may indicate inefficient promoter usage. Therefore the Ad40 ElB transcriptional regulatory region in particular the Spl binding site was studied by gel retardation assay. The ability of Ad40 and Ad2 GC box sequences (Spl binding sites) to bind either partially purified Spl or proteins from INT407 and HeLa extracts were compared. The Ad40 Spl binding site bound the Spl transcription factor but with lower affinity than the Ad2