Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses.

Simian retroviruses (SRVs), the etiological agent of a spontaneous Simian acquired immunodeficiency syndrome, endemically infects large percentages of Asian macaques housed in biomedical research colonies and severely compromises the effective use of these species as a viable research animal. We recently described the molecular cloning of a serogroup 2 SRV, D2/RHE/OR, which causes mild immunosuppression in rhesus macaques. A restriction site variant, D2/RHE/OR/V1, has also been recovered from severely ill animals endemically infected with D2/RHE/OR. We now report the complete nucleotide sequences of D2/RHE/OR and D2/RHE/OR/V1. Both infectious molecular clones retain the genetic structure typical of type D SRVs (5' LTR-gag-prt-pol-env-3'LTR) and encode identically sized 8105-bp proviruses. D2/RHE/OR and D2/RHE/OR/V1 are 99.3% similar at the amino acid level, exhibiting only 17 residue differences, of which 10 are located in the envelope glycoproteins. The molecular clones and reciprocal chimeric viruses were used to assess the contribution of different genetic domains to virus infectivity in a T cell infection assay. These experiments indicate that D2/RHE/OR has a reduced ability to infect specific T cell lines, especially Hut-78 and MT-4 cells, and that the envelope gene is not the sole determinant of in vitro tropism.

Simian AIDS type D serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3' long terminal repeat.

We describe the molecular cloning of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR) and present the sequence of its envelope (env) glycoprotein gene and 3' long terminal repeat region. This report documents the first infectious molecular clone of a serogroup 2 SRV and provides env sequence verification of genetic diversity among serogroup 2 SRV isolates.

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.

DNA structural alterations in the SV40 enhancer region are retained in vivo.

Psoralen accessibility in the SV40 regulatory region has been analyzed in vivo on viral minichromosomes and extracellular virus particles using a psoralen mapping technique which we have recently described. This approach takes advantage of the fact that lambda exonuclease digestion of double-stranded DNA is inhibited by the presence of 5-methylisopsoralen monoadducts. Previous analysis of purified restriction fragments from this region irradiated in vitro revealed a region of psoralen hypersensitivity which encompassed the repeated 72-base-pair (bp) enhancers and sequences 150 bp upstream toward the late region. The studies reported here demonstrate that changes in the structure of the DNA helix due to supercoiling do not affect the pattern of psoralen accessibility in this region. Additionally, no significant difference in the precise pattern of the psoralen hypersensitive domain is observed between late nucleoprotein complexes and extracellular virus. Finally, virus particles that have been through a freeze-thaw cycle, a process which we have shown eliminates the preferential photoaddition of the regulatory region in chromatin, also exhibit the same pattern of psoralen hypersensitivity.

High resolution psoralen mapping reveals an altered DNA helical structure in the SV40 regulatory region.

Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.

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.

In vitro characterization of the reaction of four psoralen derivatives with DNA.

Four psoralen derivatives were radiolabeled and used for in vitro DNA binding studies. The derivatives were compared for their dark-binding ability to DNA, photoreactivity, and for unwinding angles. The dark-binding dissociation constants we determined were 1.4 X 10(-3) M for 8-methoxypsoralen (8-MOP), 3.5 X 10(-4) M for 5-methoxypsoralen (5-MOP), and 5.5 X 10(-4) M for 5-methylisopsoralen (5-MIP). We did not detect any dark binding to DNA for 3-carbethoxypsoralen (3-CP). Photoaddition experiments indicated that the relative rates of photoaddition by psoralen to DNA (measured as psoralens bound per base pair per second) are 4.4 X 10(-5) for 5-MIP, 9.2 X 10(-6) for 5-MOP, 7.8 X 10(-6) for 8-MOP, and 4.6 X 10(-6) for 3-CP. We found the peak level of binding (for an initial base pair-to-psoralen ratio of 22) to be 27, 32.2, 31.2, and 1,538 base pairs per psoralen bound for 5-MOP, 8-MOP, 5-MIP, and 3-CP, respectively. In addition, 3-CP adducts could be photoreversed by prolonged irradiation at 360 nm. After 10 hours of irradiation, the amounts of 3-CP bound to DNA had fallen to less than 50% of the peak amount bound. In the same time, the amount of 8-MOP and 5-MOP bound had fallen to 95% of their peak values, and 5-MIP had fallen to 85% of its peak value. We also performed unwinding angle experiments to determine the amount of unwinding of the DNA helix induced per photobound derivative molecule; the unwinding angles +/- 3 degrees were 25 for 5-MOP, 28 for 8-MOP, 26 for 3-CP, and 18 for 5-MIP.

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.

Psoralen photoinactivation of herpes simplex virus: monoadduct and cross-link repair by xeroderma pigmentosum and Fanconi's anemia cells.

Furocoumarins (psoralen and its derivatives) are used to photoinactivate a variety of viruses and cell types. In the presence of long-wavelength ultraviolet light (UVA), furocoumarins bind covalently with pyrimidine residues via a cyclobutane ring. A second photoevent allows pyrimidines located on the opposite DNA strand in an adjacent base pair to react, forming a cross-link. In the experiments in this report, psoralen photoinactivation is employed to investigate human DNA repair pathways by analyzing the ability of xeroderma pigmentosum (XP) and Fanconi's anemia (FA) cells to rescue psoraleninactivated herpes simplex virus (HSV). Comparison of several XP complementation groups and one XP variant with normal human fibroblasts demonstrates that the ability of all cells to repair damage by 4,5',8-trimethylpsoralen (TMP), a derivative that forms cross-links efficiently, is similar. However, HSV photochemically reacted with 5-methylangelicin (5-MA), an isopsoralen that forms only monoadducts, is repaired at significantly lower levels in several XP complementation groups than in control fibroblast cells, which indicates that the XP repair deficiency resides in the removal of monoadducts and not of cross-links in these cell lines. Surprisingly, the FA cells rescue both TMP- and 5-MA-treated virus with slightly greater efficiency than that observed in normal human fibroblasts.

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.

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.

Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen.

Intracellular simian virus 40 (SV40) chromatin was photoreacted with a 3H-labeled psoralen derivative, hydroxymethyltrimethylpsoralen (HMT), at 48 h postinfection. Psoralen compounds have been shown to readily penetrate intact cells and, in the presence of long-wavelength UV light, form covalent adducts to DNA, preferentially at regions unprotected by nucleosomes. The average distribution pattern of [3H]HMT on the SV40 genome was determined by specific activity measurements of the DNA fragments generated by HindIII plus HpaII or by AtuI restriction enzyme digestion. At levels of 1 to 10 [3H]HMT photoadducts per SV40 molecule, the radiolabel was found to be distributed nonrandomly. Comparison of the labeling pattern in vivo with that of purified SV40 DNA labeled in vitro revealed one major difference. A region of approximately 400 base pairs, located between 0.65 and 0.73 on the physical map, was preferentially labeled under in vivo conditions. This finding strongly suggests that the highly accessible region near the origin of replication, previously observed on isolated SV40 "minichromosomes," exists on SV40 chromatin in vivo during a lytic infection.

Photoaddition of trimethylpsoralen as a probe for the intracellular organization of Escherchia coli DNA.

It has been previously demonstrated that photoreaction with 4,5',8-tri-methylpsoralen (trioxsalen) can be used as a probe for the in vivo structure of eucaryotic chromatin. We have used this probe to analyze the organization of intracellular Escherichia coli DNA. In contrast to eucaryotic DNA, bacterial DNA within the intact cell is not protected from saturating doses of trioxsalen photoaddition. The final level of covalently bound trioxsalen upon saturation is identical to that found with purified DNA. In addition, the distribution of interstrand DNA cross-links formed by low doses of trioxsalen photoadducts does not exhibit the repeating pattern that has been observed with eucaryotic nucleosomes.

Antibiotic susceptibility of beta-lactamase-producing strains of Branhamella (Neisseria) catarrhalis.

All 11 clinically significant isolates of Branhamella catarrhalis examined in this study were found to produce beta-lactamase. The enzyme was apparently not plasmid associated since extrachromosomal deoxyribonucleic acid was not detected in any of the strains. The beta-lactamase activity of all strains was significantly depressed by the beta-lactamase inhibitors clavulanic acid and CP 45899. Based on comparisons of relative susceptibility to various beta-lactam antibiotics, it was inferred that the beta-lactamase of B. catarrhalis was significantly more active against penicillin congeners than against cephalosporin congeners. Most strains were not inhibited by readily achievable serum concentrations of the pencillinase-sensitive penicillins, penicillin G, ampicillin, and amoxicillin. Methicillin was equally ineffective. With rare exceptions, most strains of B. catarrhalis were inhibited by achievable serum concentrations of seven cephalosporins (cephalothin, cephapirin, cephaloridine, cephalexin, cephamandole, cefaclor, and cefuroxin) and one cephamycin (cefoxitin). All strains were uniformly resistant to clindamycin but were inhibited by achievable serum concentrations of erythromycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Comparison of geometric mean minimum inhibitory concentrations of all antimicrobial agents tested suggested that B. catarrhalis was most susceptible to cefoxitin, erythromycin, and tetracycline.

Photochemical addition of the cross-linking reagent 4,5', 8-trimethylpsoralen (trioxaslen) to intracellular and viral simian virus 40 DNA-histone complexes.

We demonstrated here that 4,5', 8-trimethylpsoralen (trioxsalen) is a valuable probe for the structure of SV40 DNA-histone complexes. Trioxsalen readily penetrated intact cells and, in the presence of 340- to 380-nm light, covalently cross-linked DNA preferentially at the sites available for micrococcal nuclease digestion. Histograms of the lengths of the regions of SV40 DNA protected from cross-linking, as visualized by electron microscopy, indicated a repeating pattern of base pairs in DNA from both infected cells and virus particles. The ability of the trioxsalen probe to act in vivo and to map the location of protected regions may provide a powerful tool for analyzing the role of nucleosomes in the structure of the virus particle and in intracellular complexes such as transcription templates and replication intermediates.