Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI.

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.     

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.     

Virus-associated RNAs of naturally occurring strains and variants of group C adenoviruses

We compared the sequences of the virus-associated (VA) RNAs of group C adenoviruses, serotypes 1, 2, 5, and 6, and of three variants of adenovirus type 2 (Ad2) selected for loss of the BamHI restriction site in the VA RNAI gene. In the naturally occurring strains. VA RNAI exists in two forms which differ by two nucleotides: one form is found in Ad2 and Ad6, and the other is found in Ad1 and Ad5. There are three sites of variation in Va RNAII, the Ad1, Ad2, and Ad5 forms each differing from Ad6 VA RNAII at one of the positions. One of the selected variants has a four-base duplication within the BamHI cleavage site, whereas the two others have acquired a VA RNAI sequence indistinguishable from that of Ad5. The findings are interpreted in terms of the secondary structures of the VA RNAs and the interrelationships among the viruses.     

Characterization of CELO Virus Proteins That Modulate the pRb/E2F Pathway

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.     

RNA 2 of tobacco rattle virus strain TCM encodes an unexpected gene.

The sequence of the 3'-terminal 1210 nucleotides of RNA 1 and the complete sequence of 3389 nucleotides of RNA 2 of tobacco rattle virus (TRV) strain TCM has been deduced. The sequence of the 3'-terminal 1099 nucleotides of RNAs 1 and 2 was found to be identical. Thus the genome of this TRV strain is partially diploid, encoding a 16K protein in both RNA 1 and RNA 2. The sequence that is unique to RNA 2 contains two open reading frames: the coat protein cistron and a cistron for a 29.1K protein, which shows no homology with the RNA 1 encoded 28.8K protein. cDNA probes corresponding to these two open reading frames cross-hybridized to pea early-browning virus RNA 2, but not to RNA 2 of five other tobraviruses tested.     

Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.     

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal.

We have previously shown that most of the defective interfering (DI) RNA of mouse hepatitis virus (MHV) are not packaged into virions. We have now identified, after 21 serial undiluted passages of MHV, a small DI RNA, DIssF, which is efficiently packaged into virions. The DIssF RNA replicated at a high efficiency on its transfection into the helper virus-infected cells. The virus released from the transfected cells interfered strongly with mRNA synthesis and growth of helper virus. cDNA cloning and sequence analysis of DIssF RNA revealed that it is 3.6 kb and consists of sequences derived from five discontinuous regions of the genome of the nondefective virus. The first four regions (domains I to IV) from the 5' end are derived from gene 1, which presumably encodes the RNA polymerase of the nondefective virus. The entire domain I (859 nucleotides) and the first 750 nucleotides of domain II are also present in a previously characterized DI RNA, DIssE, which is not efficiently packaged into virions. Furthermore, the junction between these two domains is identical between the two DI RNAs. The remaining 77 nucleotides at the 3' end of domain II and all of domains III (655 nucleotides) and IV (770 nucleotides) are not present in DIssE RNA. These four domains are derived from gene 1. In contrast, the 3'-most domain (domain V, 447 nucleotides) is derived from the 3' end of the genomic RNA and is also present in DIssE. The comparison of primary sequences and packaging properties between DIsse and DIssF RNAs suggested that domains III and IV and part of the 3' end of domain II contain the packaging signal for MHV RNA. This conclusion was confirmed by inserting these DIssF-unique sequences into a DIssE cDNA construct; the in vitro-transcribed RNA from this hybrid construct was efficiently packaged into virion particles. DIssF RNA also contains an open reading frame, which begins from domain I and ends at the 5'-end 20 bases of domain III. In vitro translation of DIssF RNA and metabolic labeling of the virus-infected cells showed that this open reading frame is indeed translated into a 75-kDa protein. The structures of both DIssE and DIssF RNAs suggest that a protein-encoding capability is a common characteristic of MHV DI RNA.