Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Factors Influencing the Enhancement of the Infectivity of Poliovirus Ribonucleic Acid by Diethylaminoethyl-Dextran

The enhancement by diethylaminoethyl-dextran (DEAE-D) of the infectivity of poliovirus ribonucleic acid (RNA) for cell cultures was demonstrated by infective-center as well as by plaque assays, both in nonprimate (L) and primate cell systems (MK, HeLa, LLC-MK(2)). The sensitivity of plaque assays was greatly improved by using a tris (hydroxymethyl)aminomethane-buffered synthetic medium (basal medium Eagle) and freshly confluent cell monolayers. Enhancement of nucleic acid infectivity was directly dependent on the molecular weight of the DEAE-D. Two observations bearing on the action of DEAE-D appeared important: ribonuclease activity was reduced by DEAE-D, and cells pretreated with DEAE-D remained susceptible to infection with RNA in isotonic medium. Appreciable susceptibility of the treated cells persisted for at least 2 hr; the susceptible state could be reversed at will by an application of heparin. Enhancement of nucleic acid infectivity was independent of an effect of DEAE-D on intact virus and agar inhibitors.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.