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Human adenoviruses can cause serious, disseminated infections in immunocompromised patients. For pediatric allogeneic stem cell transplant patients, the case fatality rate can reach 80%. Still, there is no available antiviral drug that is specifically approved by the Food and Drug Administration for the treatment of adenovirus infections. To fill this pressing medical need, we have developed NPP-669, a prodrug of cidofovir with broad activity against double-stranded DNA viruses, including adenoviruses. Here, we report on the in vivo anti-adenoviral efficacy of NPP-669. Using the immunosuppressed Syrian hamster as the model, we show that NPP-669 is highly efficacious when dosed orally at 1 mg/kg and 3 mg/kg. In a delayed administration experiment, NPP-669 was more effective than brincidofovir, a similar compound that reached Phase III clinical trials. Furthermore, parenteral administration of NPP-669 increased its efficacy approximately 10-fold compared to oral dosing without apparent toxicity, suggesting that this route may be preferable in a hospital setting. Based on these findings, we believe that NPP-669 is a promising new compound that needs to be further investigated., Competing Interests: The authors declare no conflict of interest.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import. IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy., (Copyright © 2020 American Society for Microbiology.)

Human adenovirus (HAdV) causes minor illnesses in most patients but can lead to severe disease and death in pediatric, geriatric, and immunocompromised individuals. No approved antiviral therapy currently exists for the treatment of these severe HAdV-induced diseases. In this study, we show that the pan-histone deacetylase (HDAC) inhibitor SAHA reduces HAdV-5 gene expression and DNA replication in tissue culture, ultimately decreasing virus yield from infected cells. Importantly, SAHA also reduced gene expression from more virulent and clinically relevant serotypes, including HAdV-4 and HAdV-7. In addition to SAHA, several other HDAC inhibitors (e.g., trichostatin A, apicidin, and panobinostat) also affected HAdV gene expression. We determined that loss of class I HDAC activity, mainly HDAC2, impairs efficient expression of viral genes, and that E1A physically interacts with HDAC2. Our results suggest that HDAC activity is necessary for HAdV replication, which may represent a novel pharmacological target in HAdV-induced disease. IMPORTANCE Although human adenovirus (HAdV) can cause severe diseases that can be fatal in some populations, there are no effective treatments to combat HAdV infection. In this study, we determined that the pan-histone deacetylase (HDAC) inhibitor SAHA has inhibitory activity against several clinically relevant serotypes of HAdV. This U.S. Food and Drug Administration-approved compound affects various stages of the virus lifecycle and reduces virus yield even at low concentrations. We further report that class I HDAC activity, particularly HDAC2, is required for efficient expression of viral genes during lytic infection. Investigation of the mechanism underlying SAHA-mediated suppression of HAdV gene expression and replication will enhance current knowledge of virus-cell interaction and may aid in the development of more effective antivirals with lower toxicity for the treatment of HAdV infections., (Copyright © 2019 American Society for Microbiology.)

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC 50 ) for BCV and a 1.9-fold increased EC 50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity., (Copyright © 2018 American Society for Microbiology.)

Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log(10) GC reductions and a 2.3- and 2.4-log(10) PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log(10) GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log(10) GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.

Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.

More information is needed on the disinfection efficacy of chlorine for viruses in source water. In this study, chlorine disinfection efficacy was investigated for USEPA Contaminant Candidate List viruses coxsackievirus B5 (CVB5), echovirus 1 (E1), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated groundwater source and two partially treated surface waters. Disinfection experiments using pH 7 and 8 source water were carried out in duplicate, using 0.2 and 1 mg/liter free chlorine at 5 and 15 degrees C. The efficiency factor Hom (EFH) model was used to calculate disinfectant concentration x contact time (CT) values (mg x min/liter) required to achieve 2-, 3-, and 4-log(10) reductions in viral titers. In all water types, chlorine disinfection was most effective for MNV, with 3-log(10) CT values at 5 degrees C ranging from < or = 0.020 to 0.034. Chlorine disinfection was least effective for CVB5 in all water types, with 3-log(10) CT values at 5 degrees C ranging from 2.3 to 7.9. Overall, disinfection proceeded faster at 15 degrees C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but no single source water had consistently different inactivation rates than another. CT values for CVB5 in one type of source water exceeded the recommended CT values set forth by USEPA's Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Systems using Surface Water Sources. The results of this study demonstrate that water quality plays a substantial role in the inactivation of viruses and should be considered when developing chlorination plans.

Chlorine dioxide (ClO2) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO2 multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct99.99%) at 5 degrees C were >0.77 to <1.53 mg/liter x min and >0.80 to <1.59 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C AD40 experiments, >0.49 to <0.74 mg/liter x min and <0.12 mg/liter x min Ct99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct99.99% ranges for 5 degrees C experiments were >20.20 to <30.30 mg/liter x min and >0.68 mg/liter x min for pH 6 and 8, respectively. For 15 degrees C FCV experiments, Ct99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter x min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15 degrees C than at 5 degrees C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct99.99% ranges and EFH model Ct99.99% values demonstrated that FCV is more resistant to ClO2 than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct99.99% values are higher than Ct99.99% values calculated from bench-scale experiments and from EFH model application.

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.

Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5 degrees C than at 15 degrees C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15 degrees C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5 degrees C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV.

Infection of animal cells by a number of viruses generally results in an array of metabolic defects, including inhibition of host DNA, RNA, and protein synthesis, and morphological alterations known as cytopathic effects. For adenovirus infection there is a profound loss of cell structural integrity and a marked inhibition of host protein synthesis, the latter generally assumed necessary to enhance virus production. We examined the purpose of viral inhibition of cell translation and found that it was related in part to cytopathic wasting of infected cells. We show that viral shutoff of host translation promotes destruction of the intermediate filament network, particularly cytokeratins which are proteolysed at keratins K7 and K18 by the adenovirus late-acting L3 23-kDa proteinase. We found that if adenovirus is prevented from inhibiting cell translation, the intermediate filament network remains relatively intact, keratin proteins are still synthesized, and cells possess an almost normal morphological appearance and lyse poorly, reducing the release of nascent virus particles by several hundredfold. Remarkably, in tissue culture cells the accumulation of late viral structural proteins is only marginally reduced if host translation shutoff does not occur. Thus, a surprising major function for adenovirus inhibition of cellular protein synthesis is to enhance impairment of cellular structural integrity, facilitating cell lysis and release of progeny adenovirus particles.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.

The role of topoisomerases in the replication of human adenovirus type 5 was investigated with topoisomerase inhibitors. Both topoisomerase I and topoisomerase II inhibitors blocked adenovirus replication when added at the time of infection. Both types of inhibitors induced strand cleavages at specific sites in the adenovirus early templates. The cleavage sites were mapped near the 5' and 3' ends of the genes transcribed early during infection. At late times after infection, camptothecin, a topoisomerase I inhibitor, inhibited adenovirus DNA replication and induced the formation of single- and double-stranded fragments with breakpoints located at defined regions of the viral genome. The topoisomerase II inhibitors, VP-16 (etoposide) and ellipticine, did not block adenovirus DNA replication and did not induce an appreciable amount of double-strand cleavages in the newly synthesized DNA. On the other hand, VP-16 promoted double-strand cleavages at specific sites of nonreplicating adenovirus DNA. The packaging of adenovirus DNA into virus particles, which contain supercoiled adenovirus DNA (M.-L. Wong and M.-T. Hsu, Nucleic Acids Res. 17:3535-3550, 1989), was inhibited by the topoisomerase II inhibitors. Transcription of adenovirus major late genes was inhibited by both topoisomerase I and topoisomerase II inhibitors. In addition, camptothecin caused a premature termination of major late transcription. Electron microscopic analysis showed that adenovirus templates late after infection were arranged in topologically constrained loop domains. Together, these data provide evidence for the requirement of topoisomerase activities in the replication, transcription, and packaging of the linear adenovirus genome.

The inhibition of protein synthesis by cycloheximide markedly stimulated adenovirus 2 early transcription (assayed in vivo and in vitro in nuclei) when the drug was added 3 h after infection of HeLa cells. The stimulation was not uniform but ranged from three- to eightfold for region 1A to 10- to 30-fold for region 2 of the adenovirus genome. The increase was complete by about 60 min after treatment. The stimulation reversed rapidly after cycloheximide was removed. Treatment with cycloheximide either before infection with wild-type adenovirus type 5 or at 3 h after infection with dl312, a mutant which fails to express early gene products in HeLa cells, both resulted in levels of transcription at or substantially below the control (infection with wild-type adenovirus without treatment). Therefore we conclude that cycloheximide treatment and previous early gene expression interact to yield the maximum levels of transcription.

Viral mRNA species synthesized early in adenovirus type 2 infection in the presence of cycloheximide were compared with those synthesized in the absence of drug or in the presence of the DNA synthesis inhibitor 1-beta-D-arabinofuranosylcytosine. Cycloheximide caused approximately a 10-fold stimulation in the accumulation of [3H]uridine into early viral mRNA species. The only exception was a 24s mRNA transcribed from the transforming end of the genome; in the presence of cycloheximide, accumulation of this mRNA species was stimulated no more than 2-fold. Treatment with cycloheximide also resulted in the accumulation of polyadenylated RNAs transcribed from EcoRI-C that are heterogeneous and smaller than the 20S mRNA. Other translation inhibitors were shown to have similar effects, suggesting that inhibition of protein synthesis early after infection induces alterations in the metabolism of specific RNA sequences.

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.

A cellular transcription factor, ATF, binds to a repeated element in the adenovirus early region 4 (E4) promoter. ATF also binds to other viral early promoter regions and to the cyclic AMP (cAMP) response elements of cellular genes. In this report, we demonstrate that a single ATF-binding site located immediately upstream of the E4 TATA box, between -62 and -46, mediates induction of E4 transcription by 8-bromoadenosine-3',5-cyclic monophosphate or cholera toxin in the human hepatoma cell line HepG2 and rat pheochromocytoma cell line PC12. Different ATF-binding sites in the E4 control region independently conferred cAMP inducibility on the simian virus 40 early promoter in PC12 cells. Induction of E4 expression by cAMP was also observed in virus-infected HepG2 cells. Other viral early promoter regions that contain ATF-binding sites (E1A and E2A) were also induced by cAMP in infected cells. E4 expression was activated by the E1A 13S mRNA products in HepG2 cells. E1A trans activation appears to be distinct from the cAMP response.

The inhibitory effects of 20 selected antiviral compounds on the replication of adenoviruses (types 1 to 8) in vitro were investigated. While 18 compounds were ineffective, 2 compounds, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cyclic GMP), were highly effective against all adenovirus types assayed in human embryonic fibroblast cultures. Their 50% inhibitory doses were 1.1 microgram/ml for (S)-HPMPA and 4.1 micrograms/ml for 2'-nor-cyclic GMP. They were nontoxic for the host cells at the effective antiviral doses.

Aphidicolin is a highly specific inhibitor of DNA polymerase alpha and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33 degrees C) to nonpermissive (41 degrees C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase alpha and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase alpha came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.