Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)     

The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance.

To precisely define the functional sequence of the CHO1 gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the original 4.5-kilobase (kb) CHO1 clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of cho1 mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression. Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHO1 gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opi1, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When cho1 mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in cho1 cells.     

Genome structure of avian sarcoma virus Y73 and unique sequence coding for polyprotein p90.

The genome structure of a newly isolated sarcoma virus, Y73, was studied. Y73 is a defective, potent sarcomagenic virus and contains 4.8-kilobase (kb) RNA as its genome; in contrast, helper virus associated with Y73 had 8.5-kb RNA, similar to other avian leukemia viruses. Fingerprinting analysis these RNAs demonstrated that the 4.8-kb RNA contains a specific RNA sequence of 2.5 kb, which represents the transforming gene (yas) of Y73. This specific sequence was mapped in the middle of the genome and had at both ends 1- to 1.5-kb sequences in common with Y73-associated virus RNA. This structure is very similar to those of avian and mammalian leukemia viruses. In vitro translation of the 4.8-kb RNA and the immunospecificity of the products directly demonstrated that polyprotein p90, containing p19, is a product translated from capped 4.8-kb RNA and that the specific peptide portion is coded by the yas sequence. Protein 90, which was also found in cells transformed with Y73, was suggested to be a transforming protein.     

Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra.

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.     

茶尺蠖核型多角体病毒多角体蛋白基因核苷酸序列及其在Baculoviridae中的地位(英文)

茶尺蠖核型多角体病毒（ＥｏＳＮＰＶ）基因组的ｐｏｌｈ和ｅｇｔ基因区约１４．２ｋｂ的酶切图谱被构建．ｅｇｔ基因位于ｐｏｌｈ基因上游约４．８ｋｂ处,但转录方向与ｐｏｌｈ基因相反．ＥｃｏＲⅤ－Ｌ片段ｐｏｌｈ基因及其旁侧的１１２５核苷酸序列被测定．ｐｏｌｈ基因编码区长７３８核苷酸,可编码２４６氨基酸的多肽．起始密码子ＡＴＧ上游是一个富含ＡＴ（ＡＴ占７１．２％）的启动子区,在－５２核苷酸处有杆状病毒晚期基因启动子转录起始基序ＡＴＡＡＧ．在终止密码子下游２０８核苷酸有一个ｐｏｌｙ（Ａ）信号,ＡＡＴＡＡＡ．但ＥｏＳＮＰＶｐｏｌｈ基因起始密码子ＡＴＧ相邻核苷酸序列为ＧＴＡＡＴＧＴ,其－３是个Ｇ,这与已知的１６种其它杆状病毒ｐｏｌｈ基因－３位置均是Ａ不相同．在分析了ＥｏＳＮＰＶ和ＨａＳＮＰＶ多角体蛋白基因核苷酸序列的基础上,通过ＭＡＬＩＧＮ程序,比较了目前已发表的２６种杆状病毒包涵体蛋白的序列,ＥｏＳＮＰＶ与黄杉毒蛾核型多角体病毒（ＯｐＳＮＰＶ）的同源性为最高,核苷酸序列的同源性为８３．０％,氨基酸序列达９４．７％;与其它２０种鳞翅目ＮＰＶ的同源性也很高,核苷酸序列同源性为７２．６％～８１．９％,氨基酸序列为８３．７％～９３     

Heteroduplex Analysis of the Sequence Relationships Between the Genomes of Kirsten and Harvey Sarcoma Viruses, Their Respective Parental Murine Leukemia Viruses, and the Rat Endogenous 30S RNA

The sequence relations between Kirsten murine sarcoma virus (Ki-SV), Harvey murine sarcoma virus (Ha-SV), and a rat endogenous 30S RNA were studied by electron microscope heteroduplex analysis. The sequence relationships between the sarcoma viruses and their respective parental murine leukemia viruses (Kirsten and Moloney murine leukemia viruses), as well as between the two murine leukemia viruses, were also studied. The only observed nonhomology feature of the Kirsten murine leukemia virus/Moloney murine leukemia virus heteroduplexes was a substitution loop with two arms of equal length extending from 1.80 +/- 0.18 kilobases (kb) to 2.65 +/- 0.27 kb from the 3' end of the RNA. It is believed that this feature lies in the env gene region of the viral genomes. The Ha-SV and Moloney murine leukemia virus genomes (respective lengths, 6.0 and 9.0 kb) were homologous in a 1.0 +/- 0.05-kb region at the 3' end and possibly over a 200-nucleotide region at the 5' ends; otherwise, they were nonhomologous. Ha-SV and Ki-SV (length, 7.5 kb) were homologous in the first 4.36 +/- 0.37-kb region from the 3' end and in a 0.70 +/- 0.15-kb region at the 5' end. In between, there was a nonhomology region, possibly containing a short (0.23-kb) region of partial or total homology. The heteroduplex analysis between rat endogenous 30S RNA and Ki-SV shows that there are mixed regions of sequence homology and nonhomology at both the 5' and 3' ends. However, there is a large (4-kb) region of homology between Ki-SV and the rat 30S RNA in the center of the genomes, with only a small nonhomology hairpin feature. These studies help to define the regions of homology between the Ha-SV and Ki-SV genomes with each other and with the rat endogenous 30S RNA. These regions may be related to the sarcoma genicity of the viruses. In particular, the 0.7-kb region of homology of Ha-SV with Ki-SV at the 5' ends may be related to the formation of a 21,000-dalton phosphoprotein in cells transformed by either virus.     

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region.

cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.     

S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus

The structures of murine sarcoma virus (MuSV) ts110 viral RNA and intracellular RNA present in MuSV ts110-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV ts110 viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV ts110 RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed delta 1) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and p10 coding sequences. The 3' border of the delta 1 deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5-kb MuSV ts110 RNA, the 5' border of the deleted central region (termed delta 2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV ts110 RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV ts110 RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33 degrees C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39 degrees C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33 degrees C permits the reappearance of the 3.5-kb RNA at its original level.     

Transfer vectors for maximal expression of passenger genes in the Bombyx mori nuclear polyhedrosis virus expression system

A series of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) transfer vectors has been developed containing various lengths of the polyhedrin promoter, including sequences 3' of the initiation codon. The ATG initiation codon was mutated in some of these vectors to allow for the production of authentic nonfusion proteins. The ability of the various polyhedrin promoter constructs to direct expression of foreign gene sequences was assessed using two test genes, chloramphenicol acetyl transferase (cat), and human metallothionein II. Accumulation of cat mRNA and nonfused protein was low when only polyhedrin promoter sequences to -8 (relative to the translational start site of polyhedrin mRNA) were included in the transfer vector, but cat expression was comparable with that of the wild-type polyhedrin gene when promoter sequences to +5 were present. Further addition of polyhedrin gene sequences to +26 or +94 resulted in no further increase in expression. Similar results were obtained for expression of human metallothionein II, where constructs encoding polyhedrin-metallothionein fusion proteins containing polyhedrin sequences to at least +5 resulted in high levels of mRNA and protein accumulation. The expression vectors containing the +5, +26, or +94 BmNPV polyhedrin promoter can thus be used to direct maximal levels of production of nonfused proteins (when the polyedrin ATG has been mutated) or of fusion proteins, depending on which is more suitable for a particular application. These new vectors are a useful addition to those presently available and should increase the utility of the BmNPV expression system for large-scale protein production. (c) 1993 John Wiley & Sons, Inc.