Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor.

When KB cells are incubated for 1 h with human adenovirus type 2 or type 5 (1 microgram/ml) and a conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF-PE), protein synthesis is inhibited by 80 to 90%. Under these conditions, neither adenovirus nor EGF-PE alone has any effect on host protein synthesis. Thus, adenovirus enhances the toxicity of EGF-PE. A number of antibodies to intact virus and capsid components were tested for their ability to block the enhancing activity and virus uptake. At appropriate dilutions, antibodies prepared against intact virus and penton base blocked the enhancing activity without affecting virus uptake. Antibodies against hexon and fiber blocked virus uptake and enhancing activity in parallel. These studies suggest that the penton base is important in lysis of the vesicles which contain adenovirus and EGF-PE, and this base allows virus and toxin to enter the cytoplasm.     

Uptake of duck hepatitis B virus into hepatocytes occurs by endocytosis but does not require passage of the virus through an acidic intracellular compartment.

The infectious entry pathway of duck hepatitis B virus (DHBV) was investigated with primary duck hepatocytes. Virus uptake was measured by a selective PCR technique which allows for the detection of a successful infection without the need for viral replication or gene expression. To test whether DHBV uptake occurs by endocytosis, the effects of energy depletion were analyzed. The requirement for an acidic intracellular pH was tested with the lysosomotropic agent ammonium chloride. The data show that energy depletion prevents the uptake of DHBV into primary hepatocytes whereas ammonium chloride has no effect. From these data, we conclude that DHBV is taken up by its host cells by endocytosis. However, in contrast to that of most other enveloped viruses, escape of DHBV from the endocytotic route does not depend on an acidic intracellular compartment.     

Hepatitis C virus entry depends on clathrin-mediated endocytosis

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.     

Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH

The effect of a number of drugs and culture conditions on the entry into cells of a strain of poliovirus 1 (Brunende) was tested. The cells were exposed in the dark to light-sensitive, neutral red-containing virus, in the presence of the drug to be tested. Then the cells were exposed to light, transferred to normal medium, and incubated overnight. Cytopathogenic effect was measured as inhibition of [3H]leucine incorporation. Compounds that dissipate proton gradients across membranes, like monensin, protonophores, and amines, and compounds that inhibit the acidification process, such as N,N'- dicyclohexylcarbodiimide (DCCD) and tributyltin, inhibited the entry of virus, but not virus binding. This was also the case with metabolic inhibitors that deplete cells for ATP. The same compounds also inhibited the cell-induced alteration of the virus particles. When cells with surface-bound virus were exposed to low pH, the virus entered efficiently, even in the presence of monensin and DCCD. The results indicate that acidification somehow facilitates the entry of the virus RNA into the cytosol and that under normal conditions the entry occurs from intracellular acidic vesicles.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.     

The mechanism of carrier-mediated transport of folates in BeWo cells: the involvement of heme carrier protein 1 in placental folate transport

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FRalpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCP1) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FRalpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.     

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Adenovirus-dependent changes in cell membrane permeability: role of Na+, K+-ATPase.

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K+-containing medium, maximum inhibition of release was obtained by 10(-5) M ouabain and half-maximal inhibition was achieved by about 0.5 X 10(-6)M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K+, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K+ about 50% was released. This activation by K+ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na+,K+-ATPase in KB cells, with maximum activation at 50 micrograms of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na+,K+-ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K+ was present in the medium. Under the conditions used, K+ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na+,K+-ATPase activity.     

Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts.

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.     

Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA.

Given the knowledge that replication-deficient adenoviruses can mediate the delivery of unlinked plasmid DNA into eukaryotic cells (K. Yoshimura, M. A. Rosenfeld, P. Seth, and R. G. Crystal, J. Biol. Chem. 268:2300-2303, 1993), this study focuses on the role of receptor-mediated endocytosis in this process. AdCFTR (an E1- E3- adenovirus type 5-based replication-deficient adenovirus containing the 4.5-kb human cystic fibrosis transmembrane conductance regulator cDNA) was added to Cos-7 cells together with plasmid pRSVL (containing the Rous sarcoma virus long terminal repeat promoter followed by the luciferase cDNA), and luciferase activity was quantified as a measure of the expression of the plasmid DNA. When AdCFTR was bound to Cos-7 cells at 4 degrees C and the cells were subsequently incubated at 37 degrees C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching > 10(4)-fold at 3,000 PFU per cell. AdCFTR-mediated increase in pRSVL was inhibited by addition of purified adenovirus fiber but not hexon, suggesting cell surface adenovirus receptors were involved in the cointernalization process. Cell lines with a high number of adenovirus receptors (Cos-7 and HeLa) showed significant AdCFTR-dependent pRSVL expression, while cell lines with low numbers of adenovirus receptors (NIH 3T3 and U-937) showed little. AdCFTR-mediated increase in the expression of pRSVL was prevented when AdCFTR was heat treated and exposed to antibody against adenovirus or when the cointernalization process was evaluated in the presence of chloroquine, conditions all known to prevent adenovirus-mediated disruption of endocytic vesicles. In contrast, the uptake of AdCFTR into Cos-7 cells was not affected by any of these conditions. When AdCFTR was exposed to UV light, its ability to grow in 293 cells was obviated, but AdCFTR-dependent increase in pRSVL expression was minimally reduced. Finally, empty capsids of AdCFTR were able to enhance the delivery and expression of plasmid pRSVL into Cos-7 cells, suggesting that the adenovirus genome is not required for AdCFTR-mediated plasmid cointernalization. Together, these observations suggest that the ability of a replication-deficient recombinantly adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor-mediated endocytosis pathway.     

The Effects of pH and Intraliposomal Buffer Strength on the Rate of Liposome Content Release and Intracellular Drug Delivery

Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (~pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H⁺] increases 250-fold from 4 × 10^–8 M (pH 7.4, outside the cell) to 10^–5 M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.     

Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.     

Increased mitochondrial uptake of rhodamine 123 by CDDP treatment.

Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5-fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells.     

rab5 GTPase Regulates Adenovirus Endocytosis

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.     

Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1

Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of Fc gammaRI (CD64), Fc gammaRII (CD32), and Fc gammaRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.     

Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A(1) prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.     

Origin of Thymidine Kinase in Adenovirus-Infected Human Cell Lines

Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthine-aminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase.