Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.     

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2''-3''-dideoxythymidine-5''-triphosphate on viral DNA synthesis.

An adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by sedimetation in neutral sucrose gradients. However, the replication complex still retained some alpha and gamma DNA-polymerase activity. Since this complex is inhibited by 2', 3' dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of DNA polymerase gamma, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase gamma. Uninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or DDThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase alpha is also involved in Ad DNA synthesis.     

Vesicular stomatitis virus in Drosophila melanogaster cells: lack of leader RNA transport into the nuclei and frequent abortion of the replication step.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) establishes a persistent, noncytopathic infection. No inhibition of host macromolecular synthesis occurs. We studied the synthesis of VSV plus-strand leader RNA, which may be directly involved in vertebrate host synthesis shut-off. Leader RNA accumulated in Drosophila cell cytoplasm, but in low amounts, it was either free or associated to structures larger than the leader RNA-N protein complexes found in vertebrate cells. Only a few leader RNA copies migrated into the cell nucleus; no increase of this transport was observed at any time during the virus cycle. Viral RNAs complementary to the 3' end of the genome and ranging in size from the leader to several hundred nucleotides were found to accumulate in Drosophila cell cytoplasm. Their synthesis was inhibited in the presence of cycloheximide, which blocks all protein synthesis and VSV replication. Correlation between the absence of VSV cytopathogenicity in Drosophila cells and the lack of leader RNA transport into their nuclei is discussed, as well as the possible relationship between the restriction of viral synthesis and the frequent initiation of an abortive replication step.     

Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity.     

Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication

Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed.     

Studies of the identity of the unknown in protein hydrolysates

DNA synthesis in the adenovirus DNA replication complex, containing host DNA polymerases-α and -γ, was inhibited completely by aphidicolin and by 2′,3′-dideoxythymidine triphosphate (ddTTP). Double reciprocal plots of DNA polymerase activity in the replication complex against each dNTP gave a straight line although the complex contained two species of DNA polymerase. Inhibition by aphidicolin of DNA polymerase activity was competitive with dTTP but that of purified DNA polymerase-α isolated from adenovirus infected KB cells was competitive with dCTP. The above results suggest that DNA polymerases-α and -γ are integrated in the replication complex to behave as a single enzyme.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

RNA elongation by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide.

The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell extract with an adenovirus 2 promoter sequence.