Simian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function

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.

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Tarnita

Wilkie

DeCaprio

2019

“…In addition, synthesis of the fiber capsid protein is further reduced by approximately 100-fold, purportedly by inappropriate splicing, reduced translation, and other unidentified factors (1,2,40,58). The block to productive infection can be relieved either by a helper function supplied by the carboxy-terminal fragment of SV40 large tumor antigen (15,30,37,53) or by a mutation localized to the N-terminal domain of the multifunctional adenovirus 72-kDa DNA-binding protein, whose carboxy-terminal domain, reminiscent of T antigen, is responsible for DNA binding, replication, transcriptional autoregulation, enhanced transformation, and assembly of adenovirus (10,41). In the presence of either of these elements, attenuation of late transcription is reduced and proper processing of the fiber message is restored.…”

mentioning

confidence: 99%

Simian virus 40 large T antigen host range domain functions in virion assembly

Spence

Pipas

1994

The simian virus 40 (SV40) T antigen host range mutants d11066 and d11140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants d11066 and d11140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with d11066 and d11140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40%0 sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants d11066 and d11140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests

“…Curiously, the large T-antigens of SV40 and BK virus contain approximately 70 additional residues beyond the COOH terminus of the polyoma protein (14,26,28,34). Deletions in the SV40 genome removing parts of this additional sequence do not appear to affect the thermosta-bility of the large T-antigen, but diminish its adenovirus helper function (6,7). This suggests that these sequences interact only miniimally with those preceding them which are homologous in structure and presumably in function to the polyoma sequences.…”

mentioning

confidence: 99%

Nucleotide sequence changes in polyoma virus A gene mutants

Thomas¹,

Vollmer²,

Folk³

1981

The mutational alterations in polyoma virus mutants ts-a and ts-25E which cause their large T-antigens to be thermolabile have been identified. In ts-a, a G leads to A transition at nucleotide 2193 causes the replacement of Ala (GCT) by Thr (ACT). In ts-25E, a G leads to T transversion at nucleotide 2883 causes the replacement of Gly (GGC) by Cys (TGC). Revertants of both mutants have been isolated and shown to have the original nucleotides restored at these positions.