Molecular characterization of a piscine retrovirus, walleye dermal sarcoma virus.
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Amino Acid Sequence↗
Biomedical subjects
V M Vogt
Publications and source records attributed to V M Vogt.
Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.
We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.
Animals↗
Proteolytic activity of purified avian sarcoma and leukemia virus NC-PR protein expressed in Escherichia coli.
Processing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases. It is not known how the activity of PR is regulated so that proteolysis occurs at this time. Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme. In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen. We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag. This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR. NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified. In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared by in vitro translation. In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography. Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains. However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely. On the basis of these results, we hypothesize that the Gag protein in vivo is also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains. The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity.
Animals↗
Proteolytic processing and particle maturation.
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Amino Acid Sequence↗
Analysis of Rous sarcoma virus Gag protein by mass spectrometry indicates trimming by host exopeptidase.
We have used electrospray ionization-mass spectrometry to investigate Gag protein structure and processing in Rous sarcoma virus, the prototype of the avian sarcoma and leukemia viruses. Molecular masses determined for the mature virion proteins MA, CA, NC, and PR agree closely with those predicted by currently accepted models for their structures. However, the data for p10 imply that only about 10% of the product has the predicted mass while the remainder is missing the C-terminal methionine residue. Molecular masses also were obtained for products generated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli. The data confirm the predicted Gag cleavage sites for PR. Thus, carboxypeptidase activity appears to be responsible for generating the des-Met form of p10. The same activity may account for the small amount of the mature des-Met CA, as previously reported. Analysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cys-165. In conjunction with published characterizations of these two peptides processed from the segment of Gag between MA and p10, these data suggest trimming of p2b by an aminopeptidase. Finally, the molecular masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins.
Amino Acid Sequence↗
Two group I ribozymes with different functions in a nuclear rDNA intron.
DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI.
Base Sequence↗
Proteolytic processing of particle-associated retroviral polyproteins by homologous and heterologous viral proteinases.
Retroviral proteinase(PR)-catalyzed cleavage of the viral Gag and Gag-Pol polyproteins within the nascent virus particle is required for productive viral infection. Kinetic characterization and specificity analyses have been reported for several retroviral PR using oligopeptide substrates. In this study, we performed a comparative analysis of PR from avian, bovine, simian and human retroviruses using polyproteins of human immunodeficiency virus (HIV) type 1 or avian leukosis virus as substrates. Polyproteins were derived from immature virus-like particles purified from culture medium of transfected or recombinant baculovirus-infected cells. Specific cleavage to the correct size intermediate and end products occurred in the presence of detergent and homologous PR. HIV-1 PR cleaved its Gag precursor to completion at a concentration of approximately 25 nM but cleaved the Gag-Pol precursor incompletely even at fourfold higher PR concentration. In contrast to the requirement for high ionic strength for peptide cleavage reported previously, we found that Gag protein cleavage by HIV-1 PR proceeded best at low ionic strength, for both of the protein substrates tested. HIV-2 PR was approximately sixfold less active than HIV-1 PR. PR from avian myeloblastosis-associated virus (MAV) yielded efficient cleavage of the HIV-1 polyprotein only at concentrations above 1 microM. Both enzymes were stimulated by high salt and their cleavage products were identical or very similar to those of HIV-1 PR. A mutant of MAV PR engineered to cleave HIV-1 peptide substrates did not cleave the HIV-1 polyprotein at a concentration of 0.4 microM. The PR of Mason Pfizer monkey virus cleaved this polyprotein very poorly, whereas PR of bovine leukemia virus cleaved it, albeit at different sites.
Alpharetrovirus↗
Differential proteolytic processing leads to multiple forms of the CA protein in avian sarcoma and leukemia viruses.
The CA (capsid) protein of avian sarcoma and leukemia viruses occurs in multiple species. Only one form has been previously characterized biochemically. We have now determined that the mature CA protein of avian sarcoma and leukemia viruses exists as three species with different C termini, ending in amino acid residues A-476, A-478, and M-479 of the Gag precursor, respectively. These structures were deduced from a combination of cyanogen bromide peptide mapping, sequence analysis of tryptic peptides, and electrospray mass spectrometry. The three forms of CA were detected in the same ratios in Rous sarcoma virus and avian myeloblastosis virus and therefore are likely to represent a common feature of members of this genus of avian retroviruses. The only previously reported CA species, CAM-479, accounts for only about 36% of the total CA protein, while CAA-476 and CAA-478 account for 55 and 9%, respectively. From the analysis of peptides cleaved in vitro by PR, the viral protease, we infer that the cleavage site between A-476 and A-477 not only is recognized by PR but is the preferred site. We were unable to determine if A-478/A-479 is a cleavage site for PR or alternatively if CAA-478 results from further processing of CAM-479 by a carboxypeptidase. To study the biological significance of residues A-477 to M-479, we constructed genetically altered viruses in which deletions removed either residues 477 to 479 or 477 to 488. The resulting virus particles appeared to assembly with normal efficiencies, but the latter mutant showed slowed proteolytic processing. Neither of the mutants was infectious.
Amino Acid Sequence↗
Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1.
The internal structural proteins of retroviruses are proteolytically processed from the Gag polyprotein, which alone is able to assemble into virus-like particles when expressed in cells. All Gag proteins contain domains corresponding to the three structural proteins MA, CA, and NC. We have expressed the CA and NC domains together as a unit in Escherichia coli, both for Rous sarcoma virus (RSV) and for human immunodeficiency virus type 1 (HIV-1). We also expressed a similar HIV-1 protein carrying the C-terminal p6 domain. RSV CA-NC, HIV-1 CA-NC, and HIV-1 CA-NC-p6 were purified in native form by classic methods. After adjustment of the pH and salt concentration, each of these proteins was found to assemble at a low level of efficiency into structures that resembled circular sheets and roughly spherical particles. The presence of RNA dramatically increased the efficiency of assembly, and in this case all three proteins formed hollow, cylindrical particles whose lengths were determined by the size of the RNA. The optimal pH at which assembly occurred was 5.5 for the RSV protein and 8.0 for the HIV-1 proteins. The treatment of the RSV CA-NC cylindrical particles with nonionic detergent, with ribonuclease, or with viral protease caused disassembly. These results suggest that RNA plays an important structural role in the virion and that it may initiate and organize the assembly process. The in vitro system described should facilitate the dissection of assembly pathways in retroviruses.
Amino Acid Sequence↗
Nucleotide sequence and protein analysis of a complex piscine retrovirus, walleye dermal sarcoma virus.
Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3' end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue. Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.
Amino Acid Sequence↗
In situ hybridization and immunohistochemical study of walleye dermal sarcoma virus (WDSV) nucleic acids and proteins in spontaneous sarcomas of adult walleyes (Stizostedion vitreum).
Twenty-two anatomically independent dermal sarcomas from six adult walleye fish (Stizostedion vitreum) collected during the spring from Oneida Lake, New York, were examined by in situ hybridization and immunohistochemistry for the presence of walleye dermal sarcoma virus (WDSV). The viral RNA, DNA, and 90-kd protein were localized at the cellular level. Riboprobes complementary to the 5' terminal region of WDSV genome were used to detect viral nucleic acids. Rabbit polyclonal antiserum was generated against the 90-kd virus-associated antigen, presumably a product of the env gene, for immunohistochemical studies. Viral transcripts were detected in the neoplastic cells of all dermal sarcomas, in which they were generally abundant. Rare mononuclear inflammatory cells and cells within the epidermis also expressed viral RNA. In all sarcomas, low to moderate levels of viral DNA were present in all neoplastic and most mononuclear inflammatory and epidermal cells. Many neoplastic cells were immunopositive for the virus-associated protein. The distribution of immunopositive neoplastic cells mimicked approximately that of cells containing viral transcripts. The number of neoplastic cells with transcripts exceeded that of cells with protein, suggesting that productively infected neoplastic cells constituted a subset of the neoplastic cells that expressed WDSV transcripts. The viral antigen was also present within many mononuclear inflammatory cells. These data suggested that 1) dermal sarcomas were associated with elevated transcriptional activity of WDSV in the neoplastic cells and 2) the cell tropism of WDSV extended beyond the mesenchymal fibroblast-like neoplastic cells and included at least mononuclear inflammatory and epidermal cells.
Animals↗
An intron in the nuclear ribosomal DNA of Didymium iridis codes for a group I ribozyme and a novel ribozyme that cooperate in self-splicing.
We have discovered a unique group I intron-like insertion (DiSSU) in the nuclear small subunit ribosomal RNA gene of the myxomycete Didymium iridis. By sequence, DiSSU consists of a group I ribozyme at the 5' end, an open reading frame (ORF) in the middle, and a novel element at the 3' end. Intron RNA self-splices in vitro to yield ten major processed RNAs, including a full-length circle. The group I ribozyme can efficiently cleave at an internal processing site, which separates the group I ribozyme from the ORF. Surprisingly, deletion that remove the entire group I ribozyme do not impair cleavage at the 3' splice site, implying that the 3' element itself is a catalytic RNA. Deletions that remove portions of the 3' element prevent utilization of the 5' splice site, suggesting that this element cooperates with the upstream group I ribozyme in splicing. DiSSU appears to be the first example for the cooperative interaction of distinct ribozymes in RNA splicing.
Amino Acid Sequence↗
Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo.
In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.
Amino Acid Sequence↗
Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants.
In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.
Avian Sarcoma Viruses↗
Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells.
Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.
Animals↗
Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site.
Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.
Amino Acid Sequence↗
A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.
Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum. As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA. The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs. We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected. The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA. However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily. These survivors are of two classes. The first comprises cells that acquired one of three types of point mutations. The second comprises cells in which Pp LSU3 became inserted into the rDNA. In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats. When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses. The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene.
Animals↗
Purification of a telomere-binding protein from Physarum polycephalum.
We have purified a telomere-binding protein (PPT) from the acellular slime mold Physarum polycephalum. As shown previously (Coren, J.S., Epstein, E.M. and Vogt, V.M. (1991) Mol. Cell. Biol. 11, 2282-2290), in vitro this protein binds specifically to the double stranded (TTAGGG)n repeats that are found at the telomeres of extrachromosomal ribosomal DNA from this organism, and also at telomeres of mammalian chromosomes. PPT was purified from Physarum nuclear extracts by heat treatment at 90 degrees C followed by heparin-agarose fractionation and gel filtration chromatography. The most purified fraction contained two major protein bands of 10 and 7 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In gel filtration chromatography PPT migrated with a Stokes radius of 1.6 nm. Along with the previously determined sedimentation coefficient of 1.2 S, this value implies a molecular weight of about 8000, making PPT the smallest known telomere-binding protein.
Animals↗