James A. Rose

Human cytomegalovirus completely helps adeno-associated virus replication

Coinfection of adeno-associated virus (AAV) with human cytomegalovirus (HCMV) strain Towne in human embryonic fibroblasts resulted in accumulation of AAV capsid antigen and production of infectious AAV with a lag of 24 hr compared to AAV replication in AAV-adenovirus coinfections. In contrast to previous observation, these findings demonstrated that HCMV is a competent helper virus for the complete replication of AAV. In addition, HCMV and AAV were synergistic in their cytopathic effects on cells, suggesting the possibility that AAV may play a role in the pathogenicity of HCMV infections.

RNA of low molecular weight in KB cells infected with adenovirus type 2

Chromatographic analysis on methylated albumin—kieselguhr columns of newly formed, radioactive RNA from KB cells infected with adenovirus type 2 revealed an unusual radioactive component which was eluted following 4 s RNA. This RNA was present predominantly in the 100,000 g ribosomal supernatant cell fraction, and was distinguished from 4 s, 16 s and 28 s RNAs on the basis of its behavior on methylated albumin columns, base composition, sedimentation velocity and behavior on Sephadex G100 columns. This RNA component has certain characteristics in common with a “5 s” RNA associated with ribosomes from uninfected KB cells and a variety of other organisms. The effect of deoxy-fluorouridine and actinomycin D on the metabolism of this unusual RNA suggests that DNA synthesis and transcription of DNA into RNA are required for its formation.

Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA

We have shown previously that replication of defective parvoviruses [adeno-associated viruses (AAV)] requires several early adenovirus (Ad) gene products [J. E. Janik, M. M. Huston, and J. A. Rose (1981) Proc. Natl. Acad. Sci. USA 78, 1925-1929]. To examine their possible roles in the transcription and translation of AAV mRNA, 293-31 cells, a human embryonic kidney cell line that constitutively expresses the Ad early region IA and IB gene products, were transfected with a pBR325 plasmid (pLH1) that contains a duplex AAV2 DNA segment (0.03-0.97 map units) which encompasses the promoters and coding sequences necessary for expression of all AAV polypeptides. When cells were transfected with pLH1 alone, both spliced and unspliced AAV-specific cytoplasmic RNAs accumulated. These transcripts were capable of directing synthesis of the three AAV capsid polypeptides in vitro, whereas in vivo synthesis of AAV protein was not detected by immunofluorescence or immunoprecipitation. When cells were cotransfected with pLH1 and intact Ad DNA, the level of cytoplasmic AAV RNA was enhanced and AAV protein was synthesized in vivo. Additional experiments demonstrated that in vivo AAV protein synthesis also could be induced when pLH1 was cotransfected with plasmids that contain the Ad DNA-binding protein (pDBP) and VA I RNA (p2BalM) genes; however, a low level of in vivo AAV capsid protein was occasionally detected in cotransfections with pLH1 and a plasmid that contains both VA I and VA II RNA coding sequences (p2SalC). Cotransfection of pLH1 and pDBP or pLH1 and p2SalC showed complex alterations in the steady-state patterns of AAV cytoplasmic transcripts. In both cases, increased levels of transcripts, particularly the 2.3-kb spliced species, were detected in comparison to levels seen in cells transfected with pLH1 alone. Despite these increases, however, there was little, if any, induction of AAV protein synthesis unless both the DNA-binding protein (DBP) and VA I RNA coding sequences were present in cotransfection with pLH1. We conclude that, in 293-31 cells, the Ad VA I RNA and DBP gene products regulate AAV capsid protein synthesis at least at two levels: (i) by increasing the steady-state levels of structural protein transcripts in the cytoplasm, especially the spliced species, and (ii) by enhancing the translation of these messages.

Adeno-associated virus DNA replication is induced by genes that are essential for HSV-1 DNA synthesis

Adeno-associated virus (AAV) DNA replication is not detectable unless cells are coinfected with a helper adenovirus (Ad) or herpesvirus or unless AAV infection is carried out in certain established cell lines that have been treated with various metabolic inhibitors or uv irradiation. In helper-dependent infections, it has been shown that AAV DNA synthesis depends on one or more early Ad genes, whereas little is known concerning any herpesvirus gene that promotes AAV DNA synthesis. In this study we tested the ability of four cloned Xbal fragments of herpes simplex virus type 1 (HSV-1) DNA to induce AAV DNA synthesis in Vero cells. Cotransfections, which were carried out with pAV1 (an infectious AAV2 plasmid), revealed that AAV DNA synthesis could be optimally induced by three of these clones (C,D, and F) plus a clone of the HSV-1 ICP4 (IE 175) gene. ICP4, an immediate early gene, was presumably required to activate expression of other HSV genes. To help identify the additionally needed HSV genes, we tested Xbal C,D, and F subclones that contain genes previously found necessary for origin-dependent HSV DNA synthesis and found that at least five of these genes (UL 5, 8, 9, 29, and 30) contributed to the induction of AAV DNA synthesis. In contrast to their absolute requirement for HSV DNA synthesis, none of these genes were strictly necessary for AAV DNA replication. Because they are all known to specify proteins that are directly involved in HSV DNA synthesis, our results suggest that some or all of their products also may directly participate in the replication of AAV DNA.

RNA Production in Adenovirus-Infected KB Cells.

Abstract Ribonucleic acid (RNA) from adenovirus type 2-infected KB cells was examined with DNA-RNA homology techniques ( Nygaard and Hall, 1963 ), sucrose gradient analysis and methylated albumin column chromatography. The presence of viral complementary RNA was detected as early as 9 hours after infection, and relatively larger amounts were present 28 hours after infection. Although sucrose gradient analysis revealed no differences in radioactive labeling of RNA from infected and uninfected KB cells, an unusual nucleic acid component appeared in RNA from infected KB cells when analysis was performed with methylated albumin column chromatography. This component was absent from uninfected cell cultures.

Specific fragmentation of adenovirus heteroduplex DNA molecules with single-strand specific nucleases of Neurospora crassa☆

Abstract Heteroduplex DNA molecules, prepared by hybridizing DNA of adenovirus serotypes 1, 2 and 5 in all three combinations, have been shown by electron microscopy to contain two specifically located mismatched regions. Single-strand specific nucleases from Neurospora crassa cleaved the heteroduplexes into three fragments as demonstrated by sucrose gradient analysis, agarose gel electrophoresis and electron microscopy. These were produced in equimolar amounts, and were shown by alkaline sucrose gradient analysis to be free of internal single-strand interruptions. The size of each of the fragments was determined by sucrose gradient analysis and by electron microscopic measurements of the contour lengths. The three values obtained correspond to the lengths of the three double-stranded regions in the untreated heteroduplex molecules. The fragments, free of detectable single-stranded tails, have been isolated preparatively from neutral sucrose gradients. Renaturation kinetic analyses support the conclusion that each fragment represents a unique segment of the adenovirus genome.

Transcription in vivo of a defective parvovirus: Sedimentation and electrophoretic analysis of RNA synthesized by adenovirus-associated virus and its helper adenovirus

Abstract We have studied viral RNA synthesized in KB cells coinfected with adenovirus-associated virus type 2 (AAV-2) and adenovirus type 2 (Ad2) as the helper. RNA was analysed in nonaqueous, denaturing solvents either by sucrose density gradient sedimentation in 99% dimethyl sulfoxide or by acrylamide gel electrophoresis in 98% formamide. The analyses revealed two populations of AAV RNA, (i) a single, discrete 20 S species and (ii) a very heterogenous population ranging in size from 4 S to 18 S. In the nucleus both populations of AAV RNA were observed but in the cytoplasm the 20 S RNA was mainly present in the polysome fraction and the heterogenous RNA was mainly in the nonpolysomal fraction. The 20 S RNA therefore appears to be the only functional AAV message species. The heterogenous AAV RNA may arise from incomplete transcription or degradation of the 20 S RNA in the nucleus or cytoplasm. The molecular weight of the 20 S AAV RNA was approximately 0.9 × 106–1.0 × 106 which accounts for the entire region of the AAV genome that is stably transcribed. This 20 S RNA may be monocistronic and thus AAV DNA may contain only one gene. The 20 S AAV RNA did not appear to be formed by posttranscriptional cleavage of a larger nuclear precursor. In the same experiments the posttranscriptional cleavage of Ad RNA was clearly observed. At least nine species of Ad RNA were present in the cytoplasm late after infection and at least six of these were associated with polysomes. Nonpolysomal, cytoplasmic Ad RNA was heterogenous but contained one discrete, 5.5 S species (VA RNA) which was transported to the cytoplasm more rapidly than either Ad or AAV message RNA or cellular rRNA.

Structural characterization of the adenovirus 18 inverted terminal repetition

Abstract Adenovirus (Ad) 18 DNA from two plaque isolates (P-1 and P-2) derived from the prototype strain has been analyzed by cleavage with restriction endonucleases and by electron microscopic heteroduplexing techniques. Fragment sizes were determined by contour length measurements, sucrose sedimentation, and agarose gel electrophoresis. A physical ordering of fragments was obtained by comparative digestion of isolated fragments and heteroduplex mapping. End fragments were identified by their susceptibility to exonuclease digestion and by the presence of covalently attached terminal proteins. It was found that P-2 DNA molecules were about 4% longer than P-1 molecules, and that the observed length difference was due to a longer inverted terminal repetition (ITR) in P-2 DNA. Both the P-1 and P-2 ITR genotypes remained unchanged after five additional virus passages. These findings indicate that infectious Ad18 genomes can carry ITRs of different length. Based on direct nucleotide sequence analysis, the P-1 ITR is 165 bases long and possesses extensive homology with the ITR of Adl2. In addition, the P1 ITR contains several short base tracts that are variably present in the ITRs of more distantly related serotypes. One of these tracts (5′…TGACGT) is also found near the ends of DNA from both autonomous and Ad-dependent (AAV) parvoviruses.

Transcription of adenovirus-associated virus RNA in isolated KB cell nuclei

Abstract Transcription of the genomes of the defective parvovirus, adenovirus-associated virus (AAV) type 2, and a helper virus, adenovirus (Ad) type 2, was studied in nuclei isolated from infected KB cells. As measured by the incorporation of [3H]UTP into acid-insoluble product, nuclei prepared 15.5 hr after coinfection with AAV and Ad (AAV-Ad nuclei) synthesized RNA for 10 min. Total RNA synthesis in AAV-Ad nuclei was greater than in uninfected nuclei but was only about 1 2 that observed in nuclei from cells infected with Ad alone (Ad nuclei). DNA-RNA hybridization with RNA samples from the AAV-Ad nuclei revealed that the in vitro synthesis of AAV-RNA (14% of total) was twice that of Ad-RNA (6% of total). When Ad or AAV-Ad nuclei were assayed at various times after infection, total RNA production was comparable until AAV transcription was well established (12 hr); thereafter, a relative depression of total RNA synthesis was observed in the AAV-Ad nuclei. However, from 12 hr on, more AAV-RNA than Ad-RNA was synthesized in AAV-Ad nuclei, and a comparison with Ad nuclei suggested a preferential inhibition of Ad-RNA transcription in the AAV-Ad nuclei. Finally, it was found that AAV, Ad, and total RNA synthesis shared the same optimum salt concentration [0.02 M (NH4)2SO4], and that 95% of Ad-RNA synthesis and greater than 99% of AAV-RNA synthesis were inhibited by levels of α-amanitin which blocked cellular RNA polymerase II activity. These data suggest that the AAV genome is also transcribed by the same polymerase that transcribes the bulk of Ad-RNA and cellular heterogeneous nuclear RNA.