Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.

Section: Discussionmentioning

confidence: 99%

Functional role of BK virus tumor antigens in transformation

Nakshatri

Pater

1988

“…It contains several discrete functional domains that contribute to the viral life cycle and to the transforming abilities of the virus, in many cases by interacting with cellular proteins and modulating their activity (11)(12)(13)(14)(15)(16)(17). The C terminus of SV40 LT (residues 627 to 708) represents a unique domain that encompasses the host range and adenovirus helper functions (18)(19)(20). Host range (HR) mutants of SV40 with specific deletions in the C terminus of LT fail to replicate efficiently and do not form plaques in restrictive cell lines, such as African green monkey kidney CV-1P cells or the human osteosarcoma cell line U-2 OS (U2OS here) (18,21,22).…”

mentioning

confidence: 99%

Contribution of DNA Replication to the FAM111A-Mediated Simian Virus 40 Host Range Phenotype

Tarnita

Wilkie

DeCaprio

2019

Host range (HR) mutants of simian virus 40 (SV40) containing mutations in the C terminus of large T antigen fail to replicate efficiently or form plaques in restrictive cell types. HR mutant viruses exhibit impairments at several stages of the viral life cycle, including early and late gene and protein expression, DNA replication, and virion assembly, although the underlying mechanism for these defects is unknown. Host protein FAM111A, whose depletion rescues early and late gene expression and plaque formation for SV40 HR viruses, has been shown to play a role in cellular DNA replication. SV40 viral DNA replication occurs in the nucleus of infected cells in viral replication centers where viral proteins and cellular replication factors localize. Here, we examined the role of viral replication center formation and DNA replication in the FAM111A-mediated HR phenotype. We found that SV40 HR virus rarely formed viral replication centers in restrictive cells, a phenotype that could be rescued by FAM111A depletion. Furthermore, while FAM111A localized to nucleoli in uninfected cells in a cell cycle-dependent manner, FAM111A relocalized to viral replication centers after infection with SV40 wild-type or HR viruses. We also found that inhibition of viral DNA replication through aphidicolin treatment or through the use of replication-defective SV40 mutants diminished the effects of FAM111A depletion on viral gene expression. These results indicate that FAM111A restricts SV40 HR viral replication center formation and that viral DNA replication contributes to the FAM111A-mediated effect on early gene expression.IMPORTANCESV40 has served as a powerful tool for understanding fundamental viral and cellular processes; however, despite extensive study, the SV40 HR mutant phenotype remains poorly understood. Mutations in the C terminus of large T antigen that disrupt binding to the host protein FAM111A render SV40 HR viruses unable to replicate in restrictive cell types. Our work reveals a defect of HR mutant viruses in the formation of viral replication centers that can be rescued by depletion of FAM111A. Furthermore, inhibition of viral DNA replication reduces the effects of FAM111A restriction on viral gene expression. Additionally, FAM111A is a poorly characterized cellular protein whose mutation leads to two severe human syndromes, Kenny-Caffey syndrome and osteocraniostenosis. Our findings regarding the role of FAM111A in restricting viral replication and its localization to nucleoli and viral replication centers provide further insight into FAM111A function that could help reveal the underlying disease-associated mechanisms.

“…These results support the suggestion that differences identified previously in the carboxy-terminal sequences of the JCV, SV40, and BKV T proteins might contribute to the different host cell specificities of the viruses (9); alterations of these sequences in SV40 have been shown recently to * Corresponding author. limit the ability of this virus to replicate in certain cells (24,34,48). This paper describes our investigation of the restricted transforming capacity of JCV in vitro.…”

mentioning

confidence: 99%

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation

Bollag

Chuke

Frisque

1989

129

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40or BKV-transformed cells. MATERIALS AND METHODSCells. Rat 2 cells (47) were cultured in Dulbecco modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), penicillin (99 U/ml), and streptomycin (73 U/ml), at 37°C in an atmosphere containing 10% CO2. 863 on August 5, 2020 by guest