Ernest Winocour

Synthesis and transmethylation of DNA in polyoma-infected cultures☆

Abstract The incorporation of label from l -methionine (methyl-H 3 ), sodium formate-C 14 , and thymidine-H 3 , into the DNA of mouse kidney cultures, labeled from the 3rd to the 36th hour after polyoma virus infection, was increased severalfold over that in the uninfected control cultures. Evidence obtained by means of several methods showed that this DNA synthesis involved cellular in addition to viral DNA. On the basis of the incorporation of the labeled methyl group from methionine into 5-methylcytosine in the DNA from both the infected and uninfected cultures, it was concluded that DNA synthesis in these cultures was accompanied by direct transmethylation. The amount of transmethylation was proportional to the amount of DNA synthesized. There was no evidence that the virus induces hypermethylation of cellular DNA. The experiments on incorporation with methionine (methyl-H 3 ) also provided evidence that the DNA extracted from polyoma virus contains 5-methylcytosine. It was calculated that the virus DNA contains approximately one-tenth the amount of 5-methylcytosine normally present in mammalian cell DNA.

Characterization of the Simian Virus 40 — specific RNA in Virus-yielding and Transformed Cells

The SV40†-specific RNA in virus-yielding and in transformed cells has been characterized by DNA-RNA hybridization and by base ratio determinations on the hybridized RNA.

High mobility group chromosomal protein 1 binds to the adeno-associated virus replication protein (Rep) and promotes Rep-mediated site-specific cleavage of DNA, ATPase activity and transcriptional repression.

High mobility group protein 1 (HMG1) is an abundant non‐histone chromosomal protein which plays a role in several nuclear events involving DNA. Here we demonstrate that HMG1 physically interacts with the human adeno‐associated virus (AAV) Rep protein. HMG1 promotes the formation of Rep–DNA complexes and stimulates the activity of Rep in site‐ and strand‐specific cleavage of DNA and the hydrolysis of ATP, functions required for viral gene regulation, replication and site‐specific integration of viral DNA into human chromosome 19. We show that HMG1 enhances Rep‐mediated repression of the AAV p5 promoter in transfected cells, suggesting that HMG1 and Rep also interact in vivo. HMG1, Rep and DNA can be immunoprecipitated as a ternary complex. Kinetic studies indicate that complexes of Rep with DNA have similar stabilities in the presence and absence of HMG1.These results suggest that the effect of HMG1 on Rep binding is exerted at the step of complex formation and thereby may reflect an activity of HMG1 in promoting the assembly of complex cellular nucleoprotein structures.

Cell-virus interactions with the polyoma virus: I. Studies on the lytic interaction in the mouse embryo system

Abstract A study of the lytic cycle of polyoma virus multiplication on mouse embryo monolayer cultures has shown that the growth curve characteristics are similar to some cell-killing viruses that do not produce tumors. In single-cycle growth experiments, progeny virus was first detected at 22 and 24 hours after infection as increases in total and free virus, respectively. After 33 hours, the increase in total virus leveled off to a plateau. The presence of viral antigen in the nuclei of infected cells, as demonstrated by fluorescent antibody staining, was first detected at the twentieth hour after infection. Following this, the proportion of cells with fluorescent viral antigen increased gradually during this single cycle of growth, thus showing the asynchrony of polyoma virus production by the cells in this system. It was shown that a large fraction of the progeny virus produced remains cell associated and that the relationship between free virus and total virus can be influenced by the concentration of horse serum in the culture medium. Although total virus production was not decreased when higher horse serum levels were incorporated into the culture medium, the fraction of total virus represented by free virus was significantly reduced. In order to convert approximately 90 % of the cells to virus production, it was found necessary to inoculate the culture with a large excess of virus (virus:cell ratio, 940:1). In cultures inoculated at virus:cell ratios of 533:1 and less, only 10 % of the cells, as measured by both infective center assay and fluorescent antibody staining, were detected as virus producers. Based upon the proportion of cells detected as virus producers, the average yields of virus were 800–1000 PFU per cell. The characteristics of the plaque assay used in these investigations, and which is based upon the lytic cycle of multiplication, have been further analyzed. In a study on the high thermostability of polyoma virus, the half-life at 37° C was found to be 7 days.

Hybridization between SV40 DNA and cellular DNA's.

Abstract In DNA-DNA hybridization tests, Simian virus 40 DNA was bound by monkey and mouse cell DNA but not by DNA from chicken livers, Escherichia coli or T4 phage. The capacity of SV40 DNA to bind to monkey cell DNA was not abolished after fractionation of the SV40 † DNA by MAK chromatography, by equilibrium centrifugation in cesium chloride—ethidium bromide density-gradients, or by band sedimentation in neutral and alkaline cesium chloride solutions. In contrast, polyoma DNA, purified by MAK chromatography, was bound to only a very small extent, if at all, by mouse cell DNA. Double labeling experiments with [ 14 C]-Br-dUrd before infection and [ 3 H]thymidine after infection showed that the apparent homology between SV40 DNA and monkey cell DNA cannot be accounted for by the presence in viral capsids of host cell DNA unlinked to viral DNA.

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.

Further studies on the incorporation of cell DNA into polyoma-related particles☆

Abstract Polyoma infection of mouse kidney cells, whose DNA was labeled with radioactive thymidine before the time of infection, resulted in the appearance of radioactive hemagglutinating particles. The radioactive DNA extracted from these particles hybridized predominantly with uninfected mouse cell DNA and displayed in CsCl solution a buoyant density (1.702 g/ml) which is characteristic of mouse DNA. The major part of the radioactive cell DNA sedimented together with the “slow” component of polyoma DNA, with an estimated sedimentation coefficient of 14.6 S. After equilibrium centrifugation in a CsCl density gradient, the radioactively labeled particles (from cells labeled prior to infection) were broadly distributed on the lighter side of the main band of plaque-forming particles, with a peak which was approximately 0.01 density units lighter. A band centrifugation method is described which moderately enhanced the separation of the lighter particles. Band centrifugation of the particles produced in cells labeled with radioactive thymidine from hour 22 to hour 70 post-infection, revealed the presence of lighter particles whose radioactive DNA hybridized with uninfected mouse cell DNA. It is concluded (1) that both the cell DNA which exists at the time of infection, as well as that made after infection, is incorporated into a minor fraction of polyoma-related, hemagglutinating particles; (2) that most of the encapsidated cell DNA, on the basis of the results of DNA-DNA hybridization tests, does not represent a highly specific segment of the cellular genome; (3) that some of the aberrant particles contain a 3 × 10 6 molecular weight fragment of cell DNA and no 20 S polyoma DNA.

On the apparent homology between DNA from polyoma virus and normal mouse synthetic RNA

Abstract Extensive purification of polyoma virions, using six different procedures, did not remove the component responsible for the observed nucleotide sequence homology between normal mouse synthetic RNA (mouse 3H-RNA) and DNA from polyoma virus. Sedimentation in a sucrose gradient of the total DNA extracted from purified polyoma virions resulted in the appearance of two sedimenting components which differed in their ability to hybridize with mouse 3H-RNA; most of the homology to mouse 3H-RNA was found to reside in the slower sedimenting component. Fractionation of heat-treated polyoma DNA by MAK 2 column chromatography produced an infectious DNA component with little or no detectable homology to mouse 3H-RNA. The significance of these findings is discussed and it is proposed that the observed homology to mouse 3H-RNA may result from the encapsidation of cellular DNA fragments during the maturation stage of virus development.

DNA Sequence Motifs Which Direct Adeno-Associated Virus Site-Specific Integration in a Model System

ABSTRACT The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288–11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966–7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.

Covalently linked cell and SV40-specific sequences in an RNA from productively infected cells

Abstract High molecular weight simian virus 40 (SV40) specific RNA molecules, whose size exceeds that of unit length virus DNA by severalfold, are present in the nuclei of productively infected monkey BS-C-1 cells. Hybridization experiments with highly purified preparations of the large viral RNA molecules show that they contain host-specific sequences covalently linked to virus-specific sequences. The results suggest that the proportion of host-specific sequences exceeds that of viral sequences in this class of viral RNA molecules and that they arise from the cotranscription of integrated viral DNA and adjacent cellular DNA during the lytic cycle of infection.