Anti-interferon activity of adenovirus-2-encoded VAI and VAII RNAs in translation in cultured human cells.

The mode of anti-interferon action of VAI and VAII RNAs of adenovirus type 2 (Ad2) was studied by transfecting interferon-alpha (IFN-alpha)-treated KB cells in culture with a plasmid construct containing the VAI or VAII RNA gene and an SV40 promoter-chloramphenicol acetyltransferase (CAT) gene construct as reporter (pSV2-CAT). The longer the treatment of KB cells with IFN-alpha (2,000 IU/ml) lasted, the higher was the inhibition of CAT expression. A maximum of 76% inhibition was attained without pronounced cytotoxicity during 48 h of treatment. The earlier the VAI RNA gene was transfected, the higher was the enhancement of CAT expression. CAT activity increased from 113 to 157% in normal cells and 200-400% in IFN-alpha treated cells, as compared with the corresponding controls without VAI RNA transfection. The level of CAT mRNA was neither appreciably decreased by IFN-alpha treatment, nor detectably increased by VAI or VAII RNA. The effect of VA RNA thus appeared to be on translation rather than on transcription. The relative constancy of the level of CAT mRNA indicated that IFN-alpha inhibition of CAT expression was not due to the activation of RNase L, but due mainly to translational repression. The level of VAII RNA expressed was only 9-13% of that of VAI RNA. Nevertheless, VAII RNA gene transfection stimulated CAT activity to 112% of the control in non-IFN-alpha-treated cells, and 126-182% in IFN-alpha-inhibited cells. When IFN-alpha treatment was started late after VAI RNA cotransfection, CAT expression increased to 169% which was higher than the expression in cotransfected control cells without IFN-alpha treatment. The enhanced level of CAT activity was in remarkable contrast to the IFN-alpha inhibited level of 25% without VA RNA co-transfection when IFN-alpha was added upon seeding. The enhanced CAT activity in cells treated late with IFN-alpha could be ascribed to higher levels of VA RNAs.

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons.

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.

The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence.

The de novo methylation of foreign DNA integrated into the mammalian genome is a fundamental process whose mechanism has not yet been elucidated. We have studied de novo methylation in adenovirus type 12 (Ad12) genomes inserted into the genomes of Ad12-induced hamster tumor cells. De novo methylation of Ad12 DNA, which is not methylated in the virion, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultivation of cloned Ad12-induced hamster tumor cell lines, the same segments in integrated Ad12 DNA in different cell lines become methylated or remain unmethylated, depending on their positions in the viral genome. (ii) When Ad12 DNA or Ad12 DNA fragments are transfected into hamster cells and permanent cell lines are established by selection for the cotransfected neomycin phosphotransferase gene, patterns of de novo methylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines. (iii) A detailed study on the topology of the integrated viral genomes in the Ad12-transformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of the integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearranged. An originally internally located Ad12 DNA segment has become transposed to the left-terminal sequences of the viral genome in several cell lines and tumors. In the complete Ad12 genomes, the internally located PstI-D fragment becomes extensively methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences. When this DNA segment has been juxtaposed to the left-terminal, hypomethylated fragment of Ad12 DNA in rearranged genomes, the PstI-D fragment remains unmethylated. We therefore reason that the initiation of de novo methylation in integrated Ad12 DNA cannot be directed exclusively by the nucleotide sequence. Other parameters, such as site of integration, conformation of integrates, mode of cell selection, or chromatin structure related to transcriptional activity, may play decisive roles.

Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome.

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA homologies between the genomes of Choristoneura fumiferana and Autographa californica nuclear polyhedrosis viruses.

The DNA sequence homology between the genomes of Choristoneura fumiferana and Autographa californica multicapsid nuclear polyhedrosis viruses (CfMNPV and AcMNPV) were compared by hybridization of nick-translated [32P]CfMNP[V DNA to restricted AcMNPV genome. In the presence of 5 x SSC and 50% formamide the CfMNPV DNA exhibited extensive homology to the AcMNPV genome. When the stringency conditions of hybridization were lowered, we observed hybridization to almost all the EcoRI fragments of AcMNPV. We then utilized the cloned EcoRI fragments from both genomes to obtain more detailed information, and to localize the hybridizing fragments on the EcoRI physical map of AcMNPV. It was clear that some CfMNPV clones hybridized to more than one fragment of the AcMNPV genome indicating that there has been some DNA sequence rearrangement in the AcMNPV genome.

Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells.

Autographa californica nuclear polyhedrosis virus (AcNPV) is one of the most extensively studied baculoviruses. We have investigated whether AcNPV or its DNA can replicate and/or persist in cultures of mammalian cells. Human HeLa cells or primary human embryonic kidney cells, simian CV1 cells, hamster BHK21 (B3) cells or Muntiacus muntjak cells growing in monolayer cultures were used in these studies. Cells were inoculated with AcNPV at multiplicities ranging from 0.1 to 100 PFU/cell. Subsequently, the inoculated cells were investigated for virus production and for the replication and the persistence of viral DNA. Extracts of inoculated cells were also screened for the occurrence of AcNPV-specific RNA. AcNPV does not multiply in any of the cell lines studied. Viral DNA replication or transcription could not be detected by blotting and nucleic acid hybridization experiments using nick-translated, cloned viral probes. Furthermore, there was no evidence for the persistence of viral DNA or of fragments of viral DNA in mass cultures of mammalian cells. A puzzling homology between pBR322 plasmid DNA and human, simian, and hamster DNAs was detected. Since mammalian cells can take up and integrate any foreign DNA at very low frequency, it cannot be ruled out by the approach chosen that a very small number of cells might have incorporated and fixed viral DNA in their genomes. As this caveat is always pertinent for any population of cells exposed to foreign DNA, this reservation does not appear to be of particular significance in safety considerations when working with baculoviruses or any virus for that matter.

Infectious DNA from Autographa californica nuclear polyhedrosis virus.

Cells from the lepidopteran Spodoptera frugiperda can be successfully transfected in culture with the DNA from Autographa californica nuclear polyhedrosis virus (AcNPV). The calcium chloride precipitation procedure has been used in conjunction with dimethyl sulfoxide treatment of the transfected cells. The highest specific infectivity observed was 6.1 x 10(4) PFU/mug of AcNPV DNA. As judged from a comparison of the restriction patterns of viral DNA preparations, the virus produced in transfection experiments was identical to the virions from which the DNA for transfection was prepared. The transfection procedure described will be useful for the genetic analysis of AcNPV DNA.

Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus I. Synthesis of intracellular proteins after virus infection.

The replication of Autographa californica nuclear polyhedrosis virus (AcNPV) in Spodoptera frugiperda cells in culture has been studied with different methods. The first virus-induced polypeptides (with molecular weights of 46K, 30K, 29K) in infected cells appeared at 3 hr postinfection. Viral DNA synthesis started at about 5 hr postinfection. By electron microscopy, intranuclear nucleocapsids were detected at 10 hr postinfection and at about the same time, the titer of intracellular infectious particles began to rise. The pattern of viral protein synthesis was rather complex; within the first 24 hr postinfection, some 30-35 different polypeptides appeared sequentially in infected cells. Some of these polypeptides seemed to be structural proteins of the virion. The 28K polyhedrin polypeptide was synthesized originally as a precursor and was modified posttranscriptionally. Polyhedrin was synthesized until late in infection. Two distinct stages exist in AcNPV replication: (i) the rapid synthesis of AcNPV-specific nucleic acids and proteins and the assembly of nucleocapsids, some of which develop by budding to extracellular virus; (ii) intranuclear membrane synthesis, polyhedra formation, and occlusion of intranuclear, enveloped virions.

Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus II. The viral DNA and the kinetics of its replication.

The kinetics of replication of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA in Spodoptera frugiperda cells in culture were studied. Viral DNA replication started at about 5 hr postinfection, the rate of viral DNA replication reached a maximum at about 18 hr postinfection and thereafter decreased. Parental viral DNA could be detected within the first hour postinfection in the total intracellular DNA by the Southern technique. There was no evidence for the occurrence of AcNPV DNA sequences which became covalently linked to cellular DNA between 1 and 3 hr post-infection in the productive cycle. The AcNPV DNA appeared as a covalently closed circular molecule of about 92 x 10(6) daltons. The AcNPV DNA did not seem to be methylated, at least there were no 5'-CmCGG3' sequences detectable in this DNA. Restriction enzyme analysis of viral DNA preparations derived from several single plaque isolates of AcNPV as well as of DNA from different virus stocks revealed a certain heterogeneity of the AcNPV DNA.