Simian Virus 40-Host Cell Interactions II. Cytoplasmic and Nucleolar Accumulation of Simian Virus 40 Virion Protein

Chantal Widmer, James A. Robb
1974 Journal of Virology  
We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsBll. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type-and
more » ... -type-and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 ,ug/ml), RNA (actinomycin D, 5 ,ug/ml), and protein synthesis (cycloheximide, 2 x 10-4 to 10 X 10-4 M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsBl1 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsBllinfected control cells. Colchicine (10-M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsBl 1 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB1l-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsBll function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection. The molecular mechanisms underlying the containing animal viruses (e.g., Simian virus 40 cytoplasmic-nuclear transport of proteins in [SV40], polyoma, adenovirus, herpesvirus). mammalian cells are poorly understood (7). These transported virus-specific proteins in-Similar mechanisms are probably used to trans-clude the tumor (T) antigens, which are early port the cytoplasmically synthesized, virus-viral functions not requiring viral DNA synthespecific proteins that are involved in the viral sis, as well as virion structural proteins, which functions and nuclear assembly of several DNAare late functions requiring viral DNA synthesis 1530 on May 9, 2020 by guest
doi:10.1128/jvi.14.6.1530-1546.1974 fatcat:oydswyh4jvcfhhoypsbdd4oz5a