The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.The ability of simian virus 40 (SV40) to regulate many aspects of the cell cycle has made it an invaluable tool for probing fundamental biological questions and the molecular origins of human cancer (13). Viral early gene products that include large T antigen (LT), small t antigen (smt), and 17k antigen are coordinately affected by N-terminal exon mutations because this exon is common to all mature spliced early genes (27, 39). Mutations in the N-terminal region of SV40 T antigens have been described that alter viral DNA replication, capsid morphogenesis, cellular transformation, immortalization, transcriptional activation, sensitization to apoptosis, and modulation of growth control signaling pathways (2,5,18,32,39). The multifunctional viral T antigens and their interactions with host proteins have been the subject of extensive recent reviews (1, 29, 39).Of particular importance to viral replication is advancing the cell cycle. Viral infection of nonpermissive cells may lead to cellular transformation because of the targeting and sustained inactivation of key regulatory proteins, including the tumor suppressor retinoblastoma (RB) family and p53. SV40 LT specifically interacts with p53, and the retinoblastoma family members pRB, p130, and p107 (39). smt enhances transformation in many cell types, particularly those that are growth arrested (39). smt specifically interacts with protein phosphatase 2A and attenuates its activity, thereby aiding the progression of S phase (43). smt alone can also transactivate the cyclin A and adenovirus E2 promoters independent of smt-dependent inactivation of protein phosphatase ...

Several studies on cross-neutralization of the lower human adenovirus types have been published (1--6). No reports, however, have been made on types 12 and 15 through 28 except those included in the original description of these types (3,7,8). In most of these papers there has been no mention of the behavior of preimmunization sera of the animals used for immunization, so that the nature of the observed cross-reactions often remains doubtful.In our laboratory rabbit immune sera have been prepared against human adenovirus types 1 through 28, and the results of the observed cross-neutralizations are reported. Some known antigenic relationships were confirmed and some others were newly established. Preimmunization sera were regularly included in tests, whenever a positive neutralization with the respective immune serum was present. Materials and MethodsViruses. Prototype strains were used for animal immunization and for neutralization tests for all types with the exception of type 12, for which a strain isolated by Dr. G. Enders-Ruckle (strain Marburg) was substituted (9). This strain yielded much higher infectivity titers than the prototype. Viruses were grown in tteLa cell cultures with a maintenance medium containing Hanks' solution, 0.50 casein hydrolysate, and 5% calf serum. Cultures showing complete cytopathic effect (CPE) were frozen and thawed three times and centrifuged ~o remove cell debris. Titrations were performed in HeLa cell culture tubes with a final reading 8 days after inoculation. Virus stocks exhibiting a titer of at least 108.5 TCIDs0 per ml., as measured by this method, were considered satisfactory for immunization. This titer level could not be obtained with type 8 and 18 virus. Consequently stocks of these viruses were concentrated 10 times by ultraeentrifugation.* Aided by grants of the Deutsche Forsehungsgemeinsehaft.

No abstract

No abstract

No abstract

No abstract