Biochemical studies on bovine adenovirus type 3. I. Purification and properties

Abstract

Bovine adenovirus type 3 (BAV3) was purified and its properties were studied. On productive infection of CKT1 cells (a cell line derived from calf kidney) with BAV3, it was observed that viral DNA synthesis was initiated after about 24 h and its rate was maximal after about 40 h. Maturation of the virus occurred several hours after this. Purified BAV3 was separated into four discrete bands by CsCl density gradient centrifugation (complete, incomplete, empty, and degraded viruses). The complete BAV3 was similar in size and structure to human and avian adenoviruses. Polyacrylamide gel electrophoresis showed that the complete BAV3 virion contained at least 10 polypeptides. The total structural proteins of the virion had a similar amino acid composition to those of human adenoviruses. DNA of the complete virus was a linear duplex and its contour length was 12.3 4 0.9 um. The S' value of the DNA was 32.9S and its buoyant density in CsCl was 1.717 g/ml. There was about 25% homology between the DNAs of BAV3 and human adenovirus type 5 by filter hybridization. It was also noted that BAV3 produced incomplete virus. The incomplete virus was similar in morphology to the complete virus and contained almost all the structural polypeptides of the latter, but lacked infectivity. However, its DNA had a deletion(s) (13%) which seemed to locate near a terminal.

Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3

“…However, BAV-3 differs from HAV-5 with respect to replication kinetics. In BAV-3 infected cells, viral DNA replication begins at about 24 h postinfection and reaches a peak after 40 h, whereas viral DNA replication in HAV-5 infected cells occurs as early as 12 h postinfection (Niiyama et al, 1975). The green fluorescent protein (GFP) expression was noticed at 12 h postinfection when the gene for GFP was placed under the control of CMV immediate early promoter in the E3 region of BAV-3 .…”

The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

“…Because the density of complete DMEM supplemented with 10% FBS is ~1.05 g/ml and the majority of infectious HCVcc wt particles have a density between 1.01 and 1.09 g/ml, we reasoned that the ability of some infectious HCVcc virions to reach their target cells in vitro is hindered because ~60% of infectious particles are less dense than the density of the standard culture medium and would thus tend to "float" rather than sink to the cell monolayer like most other viruses, whose densities are greater than 1.11 g/ml (Burtonboy et al, 1993;Hirano, Hino, and Fujiwara, 1978;Huang et al, 2004;Kokorev et al, 1976;Macnaughton and Davies, 1980;Niiyama et al, 1975;Smith et al, 1970;Weiss et al, 1996). To test the hypothesis that the relative density of HCVcc particles versus the culture medium influences the kinetics of infection in vitro, we first analyzed HCVcc RNA kinetics following infection in cDMEM supplemented with 1, 10 and 20% FBS, which correspond to media densities of 1.02, 1.05 and 1.07 g/ml, respectively (determined by calculation and empirical measurement of density).…”

“…2), which is produced in lipoprotein-deficient 293T embryonic kidney cells (Farquhar and McKeating, 2008), and ii) viral association with lipoproteins contributes to the broad density profile of HCV (Andre et al, 2002;Gastaminza, Kapadia, and Chisari, 2006;Zhong et al, 2005) (Supplemental Fig. 3A), which is unlike the majority of other viruses that exhibit uniform density (Burtonboy et al, 1993;Hirano, Hino, and Fujiwara, 1978;Huang et al, 2004;Kokorev et al, 1976;Macnaughton and Davies, 1980;Niiyama et al, 1975;Smith et al, 1970;Weiss et al, 1996), that the prolonged and multi-phasic rate of infection initiation observed (Fig. 1) might be directly due to HCVcc virions of differing density existing in the viral inoculum.…”

Spontaneous Production of a C‐Type RNA Virus in a Cell Line Derived from Rat Glioma

“…The AC cells, in the exponential phase of growth, pelletted by centrifugation and purified virus were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in epon. Thin sections prepared were stained with uranyl acetate and lead citrate (32). Purified virus was also negatively stained.…”