Characterization of two temperature-sensitive mutants of adenovirus type 5.

The properties of two temperature-sensitive mutants ts 18 and ts 19 of adenovirus type 5 were studied. It was demonstrated that they had a defect such that they failed to assemble virus and showed defective processing of infected cell polypeptides at the restrictive temperature. Analysis, after protease digestion, of the virions produced at the permissive temperature by SDS PAGE, and of the substrate availability of the mutants to the virus protein kinase suggested that polypeptide VI was defective in these mutants.     

High molecular weight virus DNA in KB cells infected with ts mutants of adenovirus type 2 under permissive and non-permissive conditions.

Temperature-sensitive (ts) mutants of adenovirus type 2 (Ad2), which are deficient in virus DNA synthesis at the non-permissive temperature, have been used to investigate whether virus DNA replication is required for the occurrence of high mol. wt. Ad2 DNA (greater than 100S, 50 to 90S) in human cells productively infected with Ad2. The high mol. wt. virus DNA has been previously shown to consist of virus and cellular DNA molecules covalently linked. The present data indicate that after infection with DNA-ts mutants, the production of high mol.wt. virus DNA is much less sensitive to restrictive conditions than the synthesis of unit length (34S) Ad2 DNA. This finding lends further support to the idea that the occurrence of high mol. wt. virus DNA is independent of the synthesis of unit length virus DNA.     

The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2.

The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.     

Virion heat-sensitivity of adenovirus type 5 temperature-sensitive mutants and interferon induction.

Kinetics of interferon induction with wild type adenovirus 5, and three temperature-sensitive mutants, ts 1, ts 18 and ts 19 were examined in chick embryo cells (CEC) at permissive (31 degrees C) and non-permissive (38 degrees C) temperatures. Interferon levels reached a maximum four days after infection with the wild type and all ts mutants at 31 degrees C. However, at 38 degrees C, ts 18 and ts 19 failed to induce interferon, while wild type and ts 1 induced normal levels which reached a maximum by three days after infection. Striking differences in virion heat sensitivities between wild type, ts 1, ts 18 and ts 19 at both 50 degrees C and 52 degrees C were detected. These results suggested that ts 18 and ts 19 might each have a mutation in a gene coding for a virion structural component. The production of infectious progeny by shift-up experiments on Hela cells showed that mutant ts 18 was affected immediately after shift-up at all times throughout the growth cycle. On the other hand, interferon production in CEC by mutant ts 18 was only affected by shift-up during the first five hours of incubation, which indicated involvement of an early gene function in elliciting interferon production.     

Structure and function of DNA binding proteins from revertants of adenovirus type 5 mutants with a temperature-sensitive DNA replication

H5ts107 and H5ts125 are two adenoviruses type 5 (Ad5) mutants with a temperature-sensitive DNA replication. Both mutants contain an altered gene encoding the DNA binding protein (DBP). We have established by nucleotide sequence analysis that both mutants carry exactly the same mutation in the DBP gene resulting in the substitution of a proline residue at position 413 in the wild-type DBP amino acid sequence (529 amino acid residues long) by a serine residue. Revertants of H5ts107 and H5ts125, which are temperature independent in plaque efficiency and growth in HeLa cells at 32 degrees and 39 degrees, were characterized by nucleotide sequence analysis of their DBP genes. Four types of revertants could be distinguished: revertants with the wild-type DBP amino acid sequence (type I) and, revertants carrying, in addition to the original H5ts107/H5ts125 mutation at position 413, intragenic second site mutations at position 508 histidine leads to tyrosine (type II), at position 352 glycine leads to aspartic acid (type III), and at position 347 alanine leads to proline (type IV), respectively. All intragenic second site mutations are located, together with the H5ts107/H5ts125 mutation, in the C-terminal 45-kD fragment of the adenovirus DBP molecule. This provides further evidence that this part of the DBP molecule plays an important role in viral DNA replication. Phenotypic characterization of the revertants (J.C. Nicolas, F. Suarez, A.J. Levine, and M. Girard (1981), Virology 108, 521-524; (J.C. Nicolas, D. Ingrand, P. Sarnow, and A.J. Levine (1982), Virology 122, 481-485) has shown that the second site mutations reveal additional functional domains in the DBP molecule.     

Complementation of adeno-associated virus growth with temperature-sensitive mutants of human adenovirus types 12 and 5.

Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesis, were able to support growth of adeno-associated virus type I at the non-permissive temperature.     

Differential complementation of adenovirus type 5 temperature-sensitive early mutants by adenovirus types 3 and 12

We have assayed the ability of human adenoviruses from heterologous subgroups to complement early temperature-sensitive mutants of the C group virus Ad5 by analyzing coinfections at the restrictive temperature for serotype-specific DNA and late protein synthesis. Our results indicate that the B group virus AM, does not complement either ts125 or the N complementation group of Ad5 (ts36, ts69, and ts149). In contrast, studies with Ad12, an A group virus, and these mutants, indicate a differential complementation in that Ad12 complements all of the mutants in the Ad5 N complementation group but does not complement ts125. Failure to complement the ts125 defect in coinfected cells has been shown to be due to the inability of the heterologous wt gene product to substitute for the ts125 gene product directly at the level of DNA replication and not at some earlier event in the virus growth cycle.     

Replication of adenovirus type 5 DNA in KB cells: Localization and fate of parental DNA during replication

The localization and fate of parental adenovirus type 5 (Ad5) DNA in KB cells at the time viral DNA replication occurs has been investigated by electron-microscope autoradiography and biochemical methods. At 18 hr after infection the majority of the parental Ad5 DNA molecules is present in the nucleus. About 60% of these molecules are involved in viral DNA replication by this time, but about 40% have not yet replicated at all. Electron-microscope autoradiography of cells infected with ³H-labeled Ad5 reveals that replicating parental Ad5 DNA molecules are randomly distributed throughout the nucleus, while nonreplicating viral DNA has a preference to locate in the periphery of the nucleus. Based on the experiments reported in this communication, the intranuclear fate of parental Ad5 DNA is discussed.     

Expression of tumor-specific surface antigens on cells infected with temperature-sensitive mutants of avian sarcoma virus.

Tumor-specific surface antigens (TSSA) have been found in all avian sarcoma virus-transformed cells so far examined. We have studied the expression of these antigens in cells infected with mutant viruses and in which a phenotypically normal or transformed state depends on the temperature of incubation.We found that in cells infected with these mutants, all of which are defective in the ability to induce host cell transformation at the nonpermissive temperature, there are differences in the expression of TSSA. The questions whether TSSA are viral-coded functions and whether they are directly involved in the maintenance of the transformed phenotype are discussed in the light of our results.     

Cross-reactive, cell-associated antigen on L929 cells infected with temperature-sensitive mutants of sindbis virus.

Temperature-sensitive (ts) mutants of Sindbis virus (SIN) were used to aid in the identification of alphavirus cross-reactive proteins on the surface of infected cells by antibody-dependent, complement-mediated cytolysis. Antisera prepared in rabbits against purified SIN or Semliki Forest viruses were highly cytotoxic for cells infected with wild-type SIN and for cells infected at the permissive temperature with maturation-defective, ts mutants of SIN belonging to several distinct complementation groups. When these SIN mutants were analyzed by antibody-dependent, complement-mediated cytolysis at the restrictive temperature only cells infected with the SIN mutant of complementation group E, ts20, participated in both homologous (with anti-SIN serum) and heterologous (with anti-Semliki Forest virus serum) antibody-dependent, complement-mediated cytolysis reactions. These data and the known defect of ts20 suggested that the cell-associated viral E1 glycoprotein was a functional target antigen for homologous and cross-immunoreactivity in alphavirus-infected cells. At the restrictive temperature there were quantitative differences in antibody-dependent, complement-mediated cytolysis reactivity of ts20- versus wild type-infected cells consistent with the suggestion that ts20-infected cells do not fully express all of the homologous or the cross-reactive antigenic determinants found in wild-type infection. Additional potential sites for antigenic determinants involved in alphavirus-immune cross-reactivity are discussed in relation to events in virus maturation.