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PubMed2006

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Arginine↗

PubMed1999

Cryphonectria hypovirus 3, a virus species in the family hypoviridae with a single open reading frame.

Isolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5-12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)(+) tail at the 3' end and a long sequence with several minicistrons at the 5' end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)(+)-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3.

Amino Acid Sequence↗

PubMed1995

A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies.

We identified a protein component of the intracellular mature vaccinia virion membrane that is a target of a potent neutralizing monoclonal antibody, 7D11, obtained from Alan L Schmaljohn. By immunofluorescent and electron microscopic analysis, MAb 7D11 was found to stain intracytoplasmic viral factories, virion membranes in cell sections, and the surface of negatively stained preparations of purified virions. The MAb 7D11 antigen, which is synthesized at late times in infection, has apparent molecular masses of 25 and 29 kDa under nonreducing and reducing conditions, respectively. The membrane antigen was most efficiently extracted from virions by NP40 detergent in combination with a reducing agent; in addition, the protein partitioned exclusively into the detergent phase when extracted with Triton X-114. Although the N-terminus of the immunoaffinity-purified protein was blocked, sequence analysis of trypic peptides revealed that the MAb 7D11 antigen was identical to the myristylated protein encoded by the L1R open reading frame previously described by C.A. Franke, E.M. Wilson, and D.E. Hruby (1990, J. Virol. 64, 5988-5996). Validation of this genetic assignment was provided by the ability of MAb 7D11 to immunoprecipitate a [3H]myristic acid-labeled product of the expected molecular weight from infected cells. In addition, we discovered that the previously described neutralizing monoclonal antibody 2D5 (Y. Ichihashi, T. Takahashi, and M. Oie, 1994, Virology 202, 834-843) also recognizes the L1R protein.

Antibodies, Monoclonal↗

PubMed2004

A group II intron-type open reading frame from the thermophile Bacillus (Geobacillus) stearothermophilus encodes a heat-stable reverse transcriptase.

The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degrees C.

Amino Acid Sequence↗

PubMed1983

Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Antigens, Bacterial↗

PubMed1998

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

3C Viral Proteases↗

PubMed2000

Genetic polymorphism across regions of the three open reading frames of "Norwalk-like viruses".

Genomic characterization of Norwalk-like human caliciviruses (NLVs) originating from outbreaks and sporadic cases of acute gastroenteritis has revealed surprisingly high levels of diversity, even in the RNA polymerase gene, which is anticipated to be highly conserved. Since information on antigenic relationship is limited, due to the lack of a tissue culture system for these viruses, strains mostly are described on the basis of their genetic relatedness. However, the lack of uniformly applied criteria has led to a confusing array of strains with different groups employing different names for similar genetic lineages. Our goal was to conduct a structured analysis of genomic relationships among NLV strains in an attempt to provide an interim framework for genotyping. We assembled a panel of 31 potentially distinct genogroup I (GGI) and genogroup II (GGII) NLVs that reflected the diversity seen in strains detected by our laboratories and in published sequences. Phylogenetic analysis of sequences from regions of the open reading frames (ORF) 1, 2 and 3 was performed in order to investigate genomic relationships. The strains sequenced fell into seven phylogenetic groups in GGI and at least five phylogenetic groups in GGII, based on greater than 80% nucleotide identity in the region of ORF2 encoding the N-terminus of the capsid protein, and consistent clustering with high bootstrap values irrespective of the method used. Analysis of the ORF1 and ORF3 regions supported for most strains the clustering as established for those derived from ORF2. In the ORF1 region, used by most laboratories for diagnostic RT-PCR, clustering was consistent when a putative genotype border was set at 15% nucleotide mismatches for viruses in GGI and at 10% for viruses in GGII. Two strains grouped within different clusters based on ORF1 and ORF2 indicating that recombination may have occurred. We discuss the implications of these observations for the classification and typing of NLVs.

Caliciviridae↗

PubMed1994

Varicella-zoster virus open reading frame 4 encodes a transcriptional activator that is functionally distinct from that of herpes simplex virus homology ICP27.

Varicella-zoster virus is the etiological agent of chickenpox and zoster in humans and belongs to the Alphaherpesvirinae subfamily within the family Herpesviridae. Much of the current understanding of gene regulation in alphaherpesviruses has been derived from studies of the prototype herpes simplex virus (HSV). In HSV, two virus-encoded, trans-regulatory proteins, ICP4 and ICP27, are essential for the replicative cycle of the virus. ICP4 is important in modulating HSV genes of all three kinetic classes, whereas the trans-regulatory effects of ICP27 are primarily associated with the expression of late genes. Recent evidence indicates that the trans-regulatory effects of ICP27 involve posttranscriptional processing of target gene transcripts (R. M. Sandri-Golding and G. E. Mendoza, Genes Dev. 6:848-863, 1992). The ICP27 homolog in varicella-zoster virus is a 452-amino-acid polypeptide encoded by the open reading frame 4 (ORF4) gene. Contrary to what is found with ICP27, we show that the ORF4 polypeptide is a transcriptional activator of diverse target promoters and has a critical requirement for the presence of upstream elements within these promoters to mediate its transcriptional effects. Evidence is also presented to implicate a critical role for the cysteine-rich, C-terminal region of the ORF4 polypeptide in its trans-regulatory functions. Specifically, by oligonucleotide-directed site-specific mutagenesis, we demonstrate that of 10 cysteine residues in the ORF4 polypeptide, only C-421 and C-426 are essential for transactivator function and suggest that these cysteine residues may participate in critical protein-protein interactions rather than protein-nucleic acid interactions to mediate ORF4 inducibility.

Base Sequence↗

PubMed2006

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Acanthamoeba↗

PubMed1986

Sequence of the Sendai virus L gene: open reading frames upstream of the main coding region suggest that the gene may be polycistronic.

The sequence of the L gene of Sendai virus, encompassing 6799 nucleotides, has been determined, completing the primary sequence of the entire virus genome. An open reading frame beginning at position 569 codes for a basic protein of 2048 amino acids with an estimated Mr of 231,608. No nucleotide sequence similarities with the analogous L gene of vesicular stomatitis virus were observed. However, comparison of the deduced amino acid sequences of both proteins revealed a conserved 18 amino acid sequence that may have functional significance. Two additional overlapping reading frames which precede the L protein sequence could encode proteins with MrS of 6474 and 14,026, suggesting that the gene is polycistronic.

Amino Acid Sequence↗

PubMed2022

Analysis of nested alternate open reading frames and their encoded proteins.

Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, 'alt-proteins' lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

Journal Article↗

PubMed1994

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.

Amino Acids↗

PubMed2002

Neither LAT nor open reading frame P mutations increase expression of spliced or intron-containing ICP0 transcripts in mouse ganglia latently infected with herpes simplex virus.

Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing ICP0 transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced ICP0 transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing ICP0 transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether ICP0 expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of ICP0 transcripts is inhibited by the product of open reading frame (ORF) P. A LAT deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of ICP0 transcripts. LAT deletion mutants did, however, appear to express reduced levels of intron-containing ICP0 transcripts. ORF P mutations did not alter levels of ICP0 transcripts in a manner consistent with inhibition of ICP0 splicing by ORF P. Although these results argue against antisense inhibition of ICP0 expression by LATs or inhibition of ICP0 splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing ICP0 transcripts in latently infected ganglia.

Animals↗

PubMed1996

Characterization of the functional gene encoding mouse class III alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) and an unexpressed processed pseudogene with an intact open reading frame.

Multiple forms of vertebrate alcohol dehydrogenase (ADH) have been identified, but only one form, class III ADH, has been conserved in all organisms studied. Class III ADH functions in vitro as a glutathione-dependent formaldehyde dehydrogenase, which suggests that this was the original function that drove the evolution of ADH. Genetic analysis of class III ADH in yeast supports this view, but such studies are lacking in higher eukaryotes. The mouse ADH family has been previously analyzed and it contains three forms of ADH including the class III enzyme. We have initiated a molecular genetic analysis of the mouse class III ADH gene (Adh-2) by screening a genomic library with a full-length cDNA. Two overlapping clones contained the complete Adh-2 gene composed of nine exons in a 12-kb region, with the placement of introns matching that observed in other mammalian ADH genes. In this screening, we also isolated a clone (psi Adh-2) that lacks introns and which resembles a processed pseudogene. psi Adh-2 contained 25 point mutations relative to the previously analyzed Adh-2 cDNA, but still retained an intact open reading frame. Northern blot analysis using gene-specific probes provided evidence that psi Adh-2 does not produce a mRNA in either liver or kidney, whereas Adh-2 does. The functionality of the two genes was also compared by fusion of their 5'-flanking regions to a lacZ reporter gene. Reporter gene expression following transfection into mouse F9 embryonal carcinoma cells indicated that only Adh-2 possesses promoter activity.

Aldehyde Oxidoreductases↗

PubMed2006

Detection of a group II intron without an open reading frame in the alpha-toxin gene of Clostridium perfringens isolated from a broiler chicken.

A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

Animals↗

PubMed2004

Tissue-specific translation of murine branched-chain alpha-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5'-untranslated region.

The committed step in the pathway for leucine, isoleucine, and valine catabolism is catalyzed by branched-chain alpha-ketoacid dehydrogenase (BCKD). This multienzyme complex is itself regulated through reversible subunit phosphorylation by a specific kinase (BCKD-kinase). Although BCKD is present in the mitochondria of all mammalian cells, BCKD-kinase has a tissue-specific pattern of expression. Various experimental, nutritional, and hormonal conditions have been used to alter the expression of BCKD-kinase, yet little is known regarding the regulation of basal BCKD-kinase expression under normal conditions including the mechanism of its tissue specificity in any organism. Here we use tissue-derived cultured cells to explore the mechanisms used to control BCKD-kinase expression. Whereas the amount of BCKD-kinase protein is significantly higher in mitochondria from C2C12 myotubes than in BNL Cl.2 liver cells, gene transcription and stability of BCKD-kinase mRNA share similar properties in these two cell types. Our results show that the amount of protein synthesized is regulated at the level of translation of BCKD-kinase mRNA and that an upstream open reading frame in the 5'-untranslated region of this transcript controls its translation. The location and putative 19-residue peptide are conserved in the mouse, rat, chimpanzee, and human genes. Likewise, gene structure of mouse, chimpanzee, and human BCKD-kinase is conserved, whereas the rat gene has lost intron 9.

5' Untranslated Regions↗

PubMed1998

Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene.

The recently identified human herpesvirus 8 (HHV-8, or Kaposi's sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi's sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865-1869, 1994; P. S. Moore et al., J. Virol. 70:549-558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0. 7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641-6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715-719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342-346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.

Amino Acid Sequence↗

PubMed2000

Satellite and defective RNAs of Cryphonectria hypovirus 3-grand haven 2, a virus species in the family Hypoviridae with a single open reading frame.

Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66-73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3'-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5'-terminal 1.4 kb derived from the 5'-end of dsRNA 1 and the 3'-terminal 2.2 kb from the 3'-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A)(+). The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5'-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5'-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40-70 residues.

Amino Acid Sequence↗