Sedimentation Properties of Simian Virus 40-Specific Ribonucleic Acid Present in Green Monkey Cells During Productive Infection and in Mouse Cells Undergoing Abortive Infection

The size distribution of polyribosome-associated simian virus 40 (SV40) ribonucleic acid (RNA) was examined at various times after productive infection. Eight hours after infection, virus-specific RNA was detected in the 14 to 17S region of a sucrose gradient by deoxyribonucleic acid (DNA)-RNA hybridization; RNA present in fractions sedimenting more rapidly did not react with SV40 DNA. At successively later times, SV40 RNA was detected in more rapidly sedimenting regions. By 24 hr, a portion of the SV40 RNA was detected in the 28S region, sedimenting slightly more rapidly than a MS2 RNA marker. Nuclear SV40 RNA, prepared from cells 48 hr after infection, was distributed in more rapidly sedimenting regions of the gradient, peaking at about 32 to 34S. Some nuclear virus-specific RNA could be detected in the 45 to 50S region. During the abortive infection of mouse cells, the sedimentation profile of SV40 RNA was very similar to that observed during the early phases of the lytic cycle.     

Density Heterogeneity of Simian Virus 40 Ribonucleic Acid Late After Infection of Permissive Cells

Ribonucleic acid (RNA) was isolated from CV-1 cells 44 hr after simian virus 40 (SV40) infection. The molecules containing SV40 base sequences were characterized with respect to their buoyant density distribution. The density of these molecules was compared to that of single- and double-stranded RNA synthesized by using SV40 DNA and Escherichia coli RNA polymerase. The results suggest that about 20% of the SV40 RNA in the infected cells consisted of partially double-stranded molecules.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VII. Characterization of the Simian Virus 40 RNA Species Induced by Five Nondefective Hybrid Viruses

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.