Nonviable mutants of simian virus 40 with deletions near the 3' end of gene A define a function for large T antigen required after onset of viral DNA replication

We inserted a single base pair into the center of a 27-base-pair palindrome within the replication origin of simian virus 40. The mutation did not directly alter the symmetry of the palindrome or the protein-binding sequences within the palindrome. DNA binding studies showed that subunits of the simian virus 40 A protein (T antigen) bound to each of the four recognition pentanucleotides in the origin palindrome but did so with reduced affinity in comparison with wild-type origins. The mutant origin cloned in a plasmid DNA failed to replicate in COS cells. Thus, precise spatial interactions among subunits of A protein are necessary for stable origin binding and are crucial for subsequent steps in the initiation of DNA replication. Furthermore, any possible functional interactions of the simian virus 40 A protein with cellular DNA would require a great fidelity of protein binding arrangements to initiate cellular DNA replication.

Section: Methodsmentioning

confidence: 99%

“…The elongated arrows below the recognition sequences represent our interpretation of the DNase protection results. Each arrow corresponds to the DNase protection domain of a subunit of protein bound to a single pentanucleotide (32). See legend to Fig.…”

Section: Methodsmentioning

confidence: 99%

Critical spatial requirement within the origin of simian virus 40 DNA replication

Cohen¹,

Wright²,

DeLucia³

et al. 1984

“…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). Under restrictive conditions, host range mutant viruses exhibit impairment at multiple stages of the viral life cycle, including decreased early (LT) and late (VP1 and VP3) gene and protein expression (22)(23)(24), impaired viral DNA replication (18), and defective virion assembly (25).…”

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.

“…More recently, the C terminus of T antigen has been described as containing a host range activity. Whereas SV40 strains carrying deletions of the C terminus of T antigen are able to grow in BSC African green monkey kidney cells, they are unable to grow in CV1 monkey kidney cells (50,64,65). Moreover, growth in permissive cells is temperature sensitive: the mutant viruses grow in BSC cells at 37°C but do not grow in this line at 32°C (16,50).…”

mentioning

confidence: 99%

“…Compared with wild-type virus, mutant stocks give 10-fold-lower yields on BSC cells and 10,000-fold-lower yields on CV1 cells (50). Mutant viruses replicate DNA at nearly wild-type levels under nonpermissive conditions, suggesting a postreplicative block to lytic growth similar to the adenovirus helper function block (50,64 ( 1992) Virol 189 762 4d12465 -truncated at 626 -E for 627 Cole et al ( 1986) J. Virol. 57 539 C8B -aa 1-659. terminating in a nonsense mutation Manos and Gluzrran ( 1985) J Virol 53 120 Both of these lack the host range domain and would be expected to lack both host range and adenovirus helper activity.…”

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