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Imaging the location of the viral RNA synthesis ..
Coupling of RNA replication and packaging has previously been observed for poliovirus (). In this case, nonreplicating poliovirus subgenomic RNAs were not trans-encapsidated by capsid protein generated from a replicating poliovirus genome. This prompted the investigators to propose that the encapsidation of poliovirus RNAs requires a direct physical interaction between the replication complexes and the assembling capsid protein. Such a direct link between replication complexes and assembling capsid protein is unlikely for FHV because pulse-chase experiments have shown that the genomic RNAs are packaged long after they have been synthesized by the FHV polymerase (). Also, in FHV, the replication of the viral RNAs is not sufficient for packaging, based on our finding that replicating RNA1 and AUG→Stop RNA2 were not packaged by capsid protein synthesized in trans from a nonreplicating template. At the same time, the possibility of a linkage in cis between RNA packaging and translation could also be excluded because nontranslating AUG→Stop RNA2 was efficiently packaged by capsid protein made in trans from wild-type, replicating RNA2.
To visualize de novo synthesis of viral RNA, we tested the ability of purified VSV L protein to incorporate fluorescent nucleotides in vitro. Viral RNA synthesis was inhibited in reactions containing fluorescein-12-UTP, -GTP or -ATP, Alexa Fluor-488-UTP or Cy3-17-UTP or the RNA products were not fluorescent (data not shown). This result indicates that L cannot incorporate nucleotides that contain such large modifications. To test whether nucleotides with smaller modifications can be incorporated into viral RNA, we supplemented in vitro transcription reactions performed in the presence of [32P]-GTP with 5-BrUTP, and monitored the products of RNA synthesis by electrophoresis on acid-agarose gels (). As the concentration of BrUTP in the reaction increased from 0–1 mM, the overall yield of RNA decreased and the transcripts migrated with a slightly faster mobility. The altered mobility of the RNA suggests that L incorporates BrUTP into the mRNA as a similar mobility shift is observed for transcripts synthesized by T7 RNA polymerase (). The presence of BrUTP in the viral transcripts was confirmed by their selective immune precipitation with an antibody directed against bromodeoxyuridine, which failed to precipitate unmodified RNA (). The BrUTP labeled mRNAs were also retained by oligo dT chromatography, which demonstrates that the mRNAs are full-length and contain polyadenylate (). The agarose-urea gels separate products based upon their molecular weight as well as charge , which likely accounts for the observed mobility shift.
Synthesis of Avian RNA Tumor Virus Structural Proteins
BERKELEY, CA – Scientists have uncovered key new information towards understanding the crucial first step in protein synthesis, the process by which the genetic code, harbored within DNA and copied into RNA, is translated into the production of proteins. This new information also helps to explain how viruses, such as Hepatitis C, are able to highjack protein synthesis machinery in humans for their own purposes.
Biochemist Jennifer Doudna and biophysicist Eva Nogales, both of whom hold joint appointments with the Lawrence Berkeley National Laboratory (Berkeley Lab), the University of California at Berkeley, and the Howard Hughes Medical Institute (HHMI), led a study in which cryo electron microscopy (cryo-EM) was used to create a 3-D model of the protein complex called eukaryotic translation initiation factor 3 (eIF3). The model showed that the eIF3 protein complex employs the same structural mechanics in the loading of either human or viral RNA to ribosomes, the complex machinery in living cells responsible for protein synthesis.
Internal ribosome entry site - Wikipedia
The site(s) within the cytoplasm at which VSV RNA synthesis occurs and the cellular requirements for RNA synthesis remain uncertain. For rabies virus, a related member of the Rhabdoviridae, pathologic specimens of infected neuronal cells identified inclusion-like structures termed Negri bodies that contain viral nucleocapsids. This led to the suggestion that such inclusions might be sites of RNA synthesis. Subsequent studies showed that Negri body-like inclusions appear to be bona fide sites of RNA synthesis as they contain the viral N, P and L proteins necessary for RNA synthesis as well as the mRNA products of transcription , . That the inclusions may be active sites of synthesis rather than storage compartments was indicated by immune fluorescence (IF) microscopy using an antibody to bromodeoxyuridine which detected inclusions following transfection of cells with bromo UTP (BrUTP) . This suggests that the rabies polymerase incorporated BrUTP into RNA that was actively synthesized at the inclusion-like structures. In contrast to those observations for rabies virus, for VSV it was suggested that RNA synthesis occurs throughout the cytoplasm . This conclusion was also based on incorporation of BrUTP into RNA . For VSV, the presence of BrUTP labeled RNA throughout the cytoplasm could, however, reflect synthesis of RNA at specific sites followed by a subsequent distribution throughout the cytoplasm. The relationship between inclusions and viral RNA synthesis remains therefore, uncertain. In addition, although experiments performed with rabies and VSV indicate that the viral polymerase can incorporate BrUTP into viral RNA, direct biochemical evidence for this is lacking.
In the present study, working with VSV, we further probed the relationship between inclusion formation and RNA synthesis. To do this, we used recombinant viruses in which P was fused to eGFP or mRFP. We show that the P protein together with the N and L proteins are localized to inclusion-like structures in infected cells. By direct biochemical analysis of the products of RNA synthesis, we demonstrate that L incorporates BrUTP into viral mRNA in vitro as well as in cells. Imaging the location of the viral RNA synthesis machinery and the viral RNA in infected cells by fluorescent microscopy revealed that the infecting RNP can synthesize mRNA throughout the cytoplasm. Following protein synthesis, however, viral RNA synthesis appears to be restricted to inclusions. The viral mRNAs are subsequently transported away from those inclusions in a microtubule-dependent manner to facilitate translation. Our experiments show that VSV does not require a specialized site for RNA synthesis, but the viral RNA synthesis machinery is redirected to inclusions following protein synthesis.
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Glossary | Linus Pauling Institute | Oregon State University
Conversely, viral proteins might be specifically targeted to such inclusions to promote RNA synthesis and/or assembly of progeny RNPs. While the input RNP can synthesize mRNA in the absence of inclusions ( and ), the picture for genome synthesis is not certain. The kinetics with which inclusions are detected in cells () suggests that genome replication might occur in the absence of inclusion formation. We cannot, however, eliminate the possibility that smaller inclusions that are not readily visualized by our microscopy approaches are present prior to genome replication. Once inclusions are formed, they become the major sites of RNA synthesis. Although we did not formally demonstrate that RNA replication itself occurs at the inclusion, viral genomes must be present at the inclusion to provide the template for mRNA synthesis. The simplest interpretation of the data is that replication as well as transcription occurs at the inclusions.
A role for adeno-associated viral vectors in gene therapy
Detection of the first RNA synthetic events even following high multiplicity infection is challenging to visualize. All studies to date have reported on the presence of sites of RNA synthesis once replication has been established. Here we took advantage of the intrinsic properties of VSV with regard the ability to infect cells at high MOI, and inhibit all RNA synthesis other than that directed by the input genomic RNP complex. By performing infections in the presence of protein synthesis inhibitor puromycin, we show that primary viral mRNA synthesis occurs throughout the cytoplasm (). The distribution of the primary mRNAs appears unaffected by nocodazole treatment of cells (), consistent with the idea that the infecting RNP can synthesize RNA anywhere within the cytoplasm and that a specialized site is not required to compartmentalize the RNA synthesis machinery. In contrast, once viral protein synthesis occurs, RNA synthesis appears to be predominantly localized to inclusions. Although our experiments demonstrate that protein synthesis is essential for the formation of the inclusion, we cannot be certain whether this reflects a requirement for viral protein synthesis alone, or whether cellular protein synthesis might also be required. The requirement for viral protein synthesis raises the possibility that the formation of such inclusions may reflect an ability of the host cell to detect the “foreign” viral proteins, which triggers a response that results in the viral replication machinery being corralled into an inclusion-like structure. Such an idea is also compatible with the observation that inclusions are not observed until viral replication is established ().
29/08/2007 · REVIEW ARTICLE
In this study, we examined the sites of viral RNA synthesis in cells infected with VSV. By direct biochemical analysis of the products of RNA synthesis in vitro and in cells we show that VSV L incorporates BrUTP into viral mRNA. We used triple wavelength confocal microscopy to image the localization of the viral mRNA and the N, P and L proteins that are necessary for RNA synthesis. The following major conclusions are apparent from our study. The incoming viral RNP can synthesize mRNA throughout the cytoplasm of the cell. Once protein synthesis occurs, viral inclusions form that contain the viral RNA synthesis machinery and are the major sites of RNA synthesis. For efficient translation the viral mRNAs are transported away from inclusions. These findings are directly relevant to understanding whether viral derived inclusions represent preferred sites of RNA synthesis established by viruses in cells, or are instead a secondary consequence of the host response to infection.
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