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1.
Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants. 总被引:5,自引:28,他引:5
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The accompanying paper (S. Kawai and T. Koyama , J. Virol. 51:147-153, 1984) describes the isolation and biological properties of a mutant, TK15 , derived from a Rous sarcoma virus mutant, tsNY68 . The cis-acting defect of the mutant is analyzed biochemically in this paper. TK15 virions released from virus-producing 15c (+) cells were deficient in viral genomic 39S RNA, although comparable amounts of viral RNAs were transcribed in 15c (+) and tsNY68 -infected cells. Analysis of provirus DNA occurring in 15c (+) cells suggested that the mutant genome had a deletion of ca. 250 bases near the 5' end of the genome somewhere between the primer binding site and the 5' end of the gag-coding region. These findings indicate that at least part of the sequence lost in the TK15 genome is indispensable for packaging viral genomic RNA into virions. TK15 induces nonvirus -producing 15c (-) transformants at high frequency. Southern blot analysis of DNAs from those 15c (-) clone cells revealed that TK15 -derived proviruses contained various extents of internal deletions. Many 15c (-) clones had a provirus carrying only the src gene with long terminal repeat sequences at both ends. The mechanism for the segregation of 15c (-) cells is discussed. 相似文献
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Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biological properties of mutant TK15 and mutant-induced transformants 总被引:23,自引:17,他引:6
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A mutant derived from a temperature-sensitive mutant of Rous sarcoma virus ( tsNY68 ) which showed extremely low infectivity was characterized. Infection of chicken embryo fibroblast cells with the mutant, TK15 , induced two types of transformants, mutant-producing 15c (+) and nonvirus -producing 15c (-) transformants. 15c (+) cells expressed all four viral genes normally and produced a normal level of virus particles. No complementation was observed between the mutant and avian leukosis viruses. However, when 15c (+) cells were cocultured with nonvirus -producing cells transformed by Y73, a replication-defective avian sarcoma virus, a high titer of Y73 virus was recovered. From its biological properties, the mutant seemed to have a defect(s) outside the viral genes. Biochemical analysis of the TK15 mutant (T. Koyama , F. Harada, and S. Kawai , J. Virol. 51:154-162, 1984) revealed that it had a defect in packaging its own genomic RNA. During replication of TK15 virus, the TK15 mutant appeared to segregate at high frequency more defective variants that induced 15c (-) transformants, in most of which only the src gene was expressed. The mechanism for the segregation of 15c (-) transformants is discussed with respect to the defect of the mutant. 相似文献
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Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined. 相似文献
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Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis-acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans. When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5' leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs. 相似文献
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Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This implies the existence of an encapsidation or packaging signal which would be responsible for selectivity. Previously, we had identified a region of the Sindbis virus genome that interacts specifically with the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the 132-mer is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome. For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene. This is the same region that was proposed to contain the packaging signal for Semliki Forest virus genomic RNA. 相似文献
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Identification of a region in the Sindbis virus nucleocapsid protein that is involved in specificity of RNA encapsidation. 总被引:14,自引:11,他引:3
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The specific encapsidation of genomic RNA by an alphavirus requires recognition of the viral RNA by the nucleocapsid protein. In an effort to identify individual residues of the Sindbis virus nucleocapsid protein which are essential for this recognition event, a molecular genetic analysis of a domain of the protein previously suggested to be involved in RNA binding in vitro was undertaken. The experiments presented describe the generation of a panel of viruses which contain mutations in residues 97 through 111 of the nucleocapsid protein. All of the viruses generated were viable, and the results suggest that, individually, the residues mutated do not play a critical role in encapsidation. However, one mutant which had lost the ability to specifically encapsidate the genomic RNA was identified. This mutant virus, which contained a deletion of residues 97 to 106, encapsidated both the genomic RNA and the subgenomic mRNA of the virus. It is proposed that the encapsidation of this second species of RNA, which is not present in wild-type virions, is the result of the loss of a domain of the nucleocapsid protein required for specific recognition of the genomic RNA packaging signal. The results suggest that this region of the protein is important in dictating specificity in the encapsidation reaction in vivo. The isolation and preliminary characterization of two independent second-site revertants to this deletion mutant are also described. 相似文献
9.
An MΨ-Containing Heterologous RNA, but Not env mRNA, Is Efficiently Packaged into Avian Retroviral Particles
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Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs. 相似文献
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Lucia Morales Pedro A. Mateos-Gomez Carmen Capiscol Lorena del Palacio Luis Enjuanes Isabel Sola 《Journal of virology》2013,87(21):11579-11590
Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. 相似文献
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Translational stability of plant viral RNAs microinjected into living cells. Influence of a 3'-poly(A) segment 总被引:5,自引:0,他引:5
G Huez Y Cleuter C Bruck L Van Vloten-Doting R Goldbach B Verduin 《European journal of biochemistry》1983,130(1):205-209
Three different alternative structural features have been shown to be present at the 3' terminus of plant viral RNAs: (a) a poly(A) track, (b) a tRNA-like structure, (c) no special structural or sequence characteristic. We have compared the translational stability after injection into frog oocytes of a representative of each type: (a) the small genomic RNA (M-RNA) of cowpea mosaic virus (CPMV), (b) the subgenomic mRNA for coat protein (RNA 4) of brome mosaic virus (BMV), (c) the subgenomic mRNA for coat protein (RNA 4) of alfalfa mosaic virus (AIMV). It has been shown that CPMV M-RNA exhibits the highest translational stability. However, the stability of AIMV RNA 4 is remarkably high and moreover significantly higher than that of BMV RNA 4. We demonstrate that, for all three viral RNA species considered, the presence of a poly(A) segment at the 3' end of the molecules improves the translational stability. From a comparative investigation in which AIMV RNA 4 was also injected into HeLa cells, it is concluded that the stability of a given non-adenylylated mRNA depends on the nature of the cytoplastic environment. 相似文献
13.
Sun B Chen L Wei X Xiang Y Liu X Zhang X 《Biochemical and biophysical research communications》2011,(4):808-813
The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types. 相似文献
14.
The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. 总被引:33,自引:24,他引:9
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Interaction of cis-acting RNA sequences with nucleocapsid proteins is one of the critical events leading to retroviral genomic RNA packaging. We have derived a potentially stable secondary structure for the packaging signal region of human immunodeficiency virus strain IIIB, using a combination of biochemical analysis and computer modelling. This region encompasses the major splice donor (SD), which is found in a highly structured conserved stem-loop. Comparison with other published human immunodeficiency virus type 1 sequences shows almost absolute nucleotide conservation in base-paired regions required to maintain this structure. Presently and previously described packaging-defective mutants would disrupt the structure. The structure depends on base pairing between nucleotide sequences 5' of the major SD which are common to both genomic and subgenomic RNAs and sequences 3' of SD which are unique to the unspliced RNA. This may explain how in retroviruses such as Rous sarcoma virus, mutations in regions common to genomic and subgenomic RNA might prevent packaging of the unspliced mRNA by disrupting a signal structure which can exist only in the genomic RNA species. 相似文献
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Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA. 总被引:30,自引:20,他引:10
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Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen. 相似文献
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Ni N Nikolaitchik OA Dilley KA Chen J Galli A Fu W Prasad VV Ptak RG Pathak VK Hu WS 《Journal of virology》2011,85(15):7603-7612
Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1. 相似文献