Evaluation of the mutagenic effects of SV40 in mouse, hamster, and mouse-human hybrid cells

We have examined the ability of SV40 to induce changes in drug or temperature resistance in mouse, hamster, and mouse-human hybrid cells. SV40 induced a substantial increase of cells resistant to 5-bromodeoxyuridine + trifluorothymidine in Balb/c 3T3 cells and induced an increase of hybrid cells resistant to 6-thioguanine. SV40 was found to be nonmutagenic or weakly mutagenic in other test systems. The 3T3 cells were T-antigen positive, exhibited a marked reduction in TK activity, were heterogeneous for [ 3 H]BrdU incorporation by autoradiography, and exhibited instability of the drug-resistance phenotype, suggesting that SV40 may be inducing resistance by an epigenetic process. SV40-induced 6-thioguanine resistance in the hybrids appears to occur predominantly by chromosome loss.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45539/1/11188_2005_Article_BF01233058.pd

Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly.

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm

Rotavirus gene silencing by small interfering RNAs

RNA interference is an evolutionarily conserved double-stranded RNA-triggered mechanism for suppressing gene expression. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, from which the spike protein VP4 projects. Here, we show that a small interfering RNA corresponding to the VP4 gene efficiently inhibits the synthesis of this protein in virus-infected cells. A large proportion of infected cells had no detectable VP4 and the yield of viral progeny was reduced. Most of the virus particles purified from these cells were triple-layered, but lacked VP4, and were poorly infectious. We also show that VP4 might not be required for the last step of virus morphogenesis. The VP4 gene silencing was specific, since the synthesis of VP4 from rotavirus strains that differ in the target sequence was not affected. These findings offer the possibility of carrying out reverse genetics in rotaviruses