Lesley M. Hallick

Giardia lamblia: autoradiographic analysis of nuclear replication.

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.

SV40 virus particles lack a psoralen-accessible origin and contain an altered nucleoprotein structure.

The nucleoprotein structure of SV40 virions was examined by photolabeling purified virus with the radioactive psoralen derivative hydroxymethyltrimethylpsoralen (HMT). Unlike SV40 chromatin in situ, the viral origin region is not preferentially accessible to drug addition. The ratio of the distribution of radioactivity in the DNA restriction fragments of virion DNA to that of purified SV40 DNA demonstrates that the photoadducts are positioned similarly on the circular molecule in both samples. Virion purified from infected cells was also analyzed for the presence of an open region and found to exhibit the same pattern of [3H]HMT addition as mature extracellular virion. The nucleosome-free region detected at the SV40 replication origin in intracellular minichromosomes is not present in either population of intact virus particles. We also examined the level of drug addition obtained when purified virion or SV40-infected cells were treated with saturating doses of [3H]HMT. Marked differences in the plateau levels of bound drug indicate that an altered nucleoprotein structure exists in SV40 virions that does not protect the DNA from photoaddition to the same extent as do the nucleosomes of intracellular SV40 DNA.

Herpes simplex virus shedding in genital secretions.

Nine women and eight men with a history of genital herpes had cultures taken for 60 consecutive days to assess the frequency and duration of viral shedding in the absence of symptoms. Daily self-obtained specimens (cervical-vaginal swabs from women and urethral swabs from men) were submitted for viral isolation. Five of 972 samples were found to contain infectious virus; two of the positive specimens correlated with overt disease. The other three positive cultures, two from the same individual, were not clearly associated with a genital infection. Thus, the overall frequency of asymptomatic viral shedding was 0.31% and occurred in two of 17 individuals. It is concluded that prolonged continuous sampling may be necessary to assess the risk of asymptomatic shedding. Infectious virus was not present in the genital secretions of most individuals (15 of 17) in the absence of a lesion, even when cultured on a daily basis for 60 days.

Optimal conditions for titration of SV40 by the plaque assay method

The parameters of the Simian Virus 40 (SV40) plaque assay on African green monkey kidney cells were optimized for reproducibility and maximum plaquing efficiency. Plaques were visible as early as 8 days postinfection; maximum titers were obtained with a 10- to 11-day incubation period. Titers read 12-16 days postinfection were not significantly higher than those observed after 10-11 days. Adsorption volumes greater than 0.1 ml/60 mm Petri dish decreased plaque forming units (PFUs) detected. Times greater than 60 min for adsorption of virus to the cell monolayer did not significantly increase the titer; adsorption times less than 60 min resulted in decreased titers. Under standard conditions, 3 ml of overlay medium containing 0.8% agar was applied following virus adsorption and again on days 5 and 10. Concentrations of fetal calf serum (FCS) in the overlay medium of 2.5 to 7.5% gave equal plaque formation. FCS concentrations of 1 and 10% resulted in slightly decreased and increased plaquing efficiencies respectively. Of the reagents tested, agar or agarose containing overlay media produced plaques of maximum number and size. An overlay of methyl cellulose resulted in the same number of plaques, but their size was reduced by approximately 70% relative to those observed in agar; thus longer incubation times were required. Gum tragacanth overlay medium was actually inhibitory to plaque development. DEAE-dextran, dextran sulfate, or DMSO added to agar overlay medium did not enhance plaque number or size, nor did they shorten the incubation period required for their detection.

The SV40 nucleosome-free region is detected throughout the virus life cycle

The structures of SV40 intracellular chromatin complexes and of extracellular virus particles were examined by photolabeling with a radioactive psoralen derivative in order to determine the fate of the exposed origin region during the virus life cycle. We have previously shown that the origin region of intracellular SV40 chromatin is preferentially accessible to psoralen derivatives in vivo, whereas psoralen adducts are uniformly distributed when purified virus particles are photoreacted. We demonstrate here that when virion is photoreacted prior to a freeze-thaw cycle, the exposed regulatory region detected in intracellular nucleoprotein complexes is also found in mature virus particles. In contrast, if the virion is frozen and thawed prior to the photoreaction, the origin is not preferentially exposed to photoaddition. Virus particles that have not been subjected to a freeze-thaw cycle were found to exhibit preferential labeling in the origin region whether they were irradiated intracellularly, in culture medium, or following purification. Banding the virus in CsCl had no significant effect on the relative accessibility of the origin region to psorealen. Our findings indicate that the open regulatory region found on intracellular SV40 chromatin persists throughout the virus life cycle.

KINETICS OF SIMIAN VIRUS 40 AND LAMBDA INACTIVATION BY PHOTOADDITION OF PSORALEN DERIVATIVES

Abstract The kinetics of psora/en photoinactivation of two distinct DNA viruses, bacteriophage λ and the papovavirus SV40 were investigated. When λ is treated with near ultraviolet light (UVA, 320‐400 nm) and 4,5′,8‐trimethylpsoralen (TMP) at 1 μg/m/, the phage is rapidly inactivated. The survival curve exhibits a distinct shoulder indicating second or higher‐order kinetics. SV40, on the other hand, is much more resistant to psoralen photoinactivation and the survival curve is linear, reflecting first order or‘pseudo‐first order’kinetics. Two TMP derivatives with increased solubility in aqueous solutions, 4′‐aminomethyl‐TMP and 4′‐hydroxymethyl‐TMP, were similarly tested. In both virus systems, TMP was much more effective. In experiments designed to examine the role of psoralen cross‐link formation in virus inactivation, treated samples were irradiated a second time in the absence of drug. Since reirradiation causes a decline in λ infectivity as great as that observed in continuously irradiated samples, cross‐links are implicated as the primary lethal event. In the case of SV40, the results of such a protocol suggest that both monoadducts and cross‐links may be lethal or that monoadduct formation may be rate‐limiting.

THE SITE-SPECIFIC INHIBITION OF Bgl I CLEAVAGE BY PSORALEN PHOTOADDUCTS

Abstract— We have investigated the site specificity of furocoumarins by using fluorescent densitometry to examine the frequency of cleavage by the restriction enzyme Bgl I. This enzyme has an 11 base pair (bp) recognition sequence which varies slightly from site to site because it includes a 5 base pair neutral region. Cleavage at all three Bgl I recognition sites in pBR322 was inhibited by the photoaddition of the psoralen derivative 4′‐hydroxymethyl‐4,5′,8‐trimethylpsoralen (HMT) which forms both crosslinks and monoad‐ducts in a dose‐dependent manner. One site, which contains two thymidines in a crosslinkable configuration, was observed to be markedly more sensitive to HMT photoadducts. In contrast Bgl I cleavage at all sites was relatively resistant to the derivative 5‐methylisopsoralen (5‐MIP), which forms only monoadducts. When HMT‐reacted DNA was generated with widely different ratios of monoad‐ducts to crosslinks (3% and 40% crosslinks), essentially the same level and pattern of inhibition was observed in both cases. Taken together, the data imply that differences in inhibition seen at the three cutting sites of Bgl I with HMT are attributable to DNA sequence and the role it plays in adduct positioning.