Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

Regulation of the biosynthesis of subgroup C adenovirus protein IVa2

The IVa2 gene is located between 16 and 11.3 map units on the left strand of the adenovirus type 5 (Ad5) genome. The coded RNA contains an intron of 277 nucleotides. To determine whether protein IVa2 is synthetized during productive infection and to obtain an immunological reagent to study its function, we prepared antibodies directed to 414 amino acids of protein IVa2 fused to the N-terminal domain of Staphylococcus aureus protein A. Western immunoblot analysis of viral proteins demonstrates that protein IVa2 is a minor component of mature viral particles and that it is also present in assembly intermediates and young virions. Thus, contrary to a previous report (H. Persson, B. Mathisen, L. Philipson, and U. Pettersson, Virology 93:198-208, 1979), protein IVa2 is not related to the 50-kDa polypeptide, a scaffolding protein present in assembly intermediates. The biosynthesis of protein IVa2 during productive infection was examined. Time course studies using immunofluorescence analysis with polyclonal antibodies targeted to protein IVa2 revealed that this protein is first synthesized at 12 h in a few cells exhibiting very striking fluorescence. Synthesis continues until at least 24 h postinfection. When hydroxyurea is added, protein IVa2 is not detected. In cells infected with mutant H5 ts125, blocked at the nonpermissive temperature (40 degrees C) in viral DNA replication, protein IVa2 is overexpressed. These results suggest that protein IVa2 synthesis requires cellular rather than viral DNA replication. RNase protection assay results indicate that hydroxyurea inhibits protein IVa2 synthesis at the transcriptional level. Thus, overexpression of protein IVa2 in H5 ts125-infected cells may be regulated at the translational level.</jats:p

Physical mapping of adenovirus type 2 temperature-sensitive mutations by restriction endonuclease analysis of interserotypic recombinants

The genome structures of about 100 interserotypic ts recombinants produced in crosses between human adenovirus type 2 (H2) and 5 (H5) temperature-sensitive mutants were analyzed by cleavage with restriction endonucleases to determine the map coordinates of the following temperature-sensitive mutants: penton base plus fiber-defective H2 ts103, -104, and -136, assembly-defective H2 ts112, fiber-defective H2 ts125, hexon-defective H2 ts118 and -121, and DNA-negative H2 ts111. H5 ts1 (100 K defective), H5 ts36 (DNA negative), H5 ts125 (mutated in the early 72,000-dalton protein), H5 ts22 (fiber defective), H5 ts58 (IIIa defective), and H5 ts18 and -19 were used as one of the parents. The physical locations of the H2 temperature-sensitive mutations thus defined are discussed in relation to the genetic map, the biological function altered, and the positions of the structural genes on the genome.</jats:p

Effects of novobiocin on adenovirus DNA synthesis and encapsidation.

Novobiocin, an inhibitor of DNA gyrase implicated in bacterial and likely mammalian, chromosome replication, inhibited the initiation, but not the elongation of human adenovirus DNA replicative synthesis. The inhibition was partially reversible, even in the presence of protein synthesis inhibitor. Novobiocin inhibited also the encapsidation of viral DNA, and this effect was independent of the block in DNA replication. It was suggested that novobiocin acted on two different functions, one involved in viral DNA replication initiation, the other in DNA encapsidation