Solved: Cellular location of viral rna synthesis, Biology
RNA viruses that replicate within the cytoplasm often form specialized structures that are the sites of RNA replication . For positive-strand RNA viruses, replication occurs on cellular membranes, including those of the endoplasmic reticulum, secretory pathway, mitochondria and other organelles –. Experiments with poliovirus and with flock house virus (FHV) have provided compelling evidence that the viral RNA and the non-structural proteins required for RNA replication are localized to such sites. For FHV, electron microscopy and tomographic reconstructions of spherule-like structures invaginated from mitochondrial membranes confirm that they contain the viral replication machinery . Double-strand RNA viruses form phase-dense inclusions or “viral factories” to which transcription competent viral cores and the machinery required for RNA synthesis are localized . In contrast to the structures formed by positive-strand RNA viruses, the double-strand RNA virus factories are not membrane bound –. The formation of such specialized replication compartments is thought to concentrate the viral machinery necessary for RNA synthesis and thereby favor catalysis. Compartmentalization of the replication machinery might also shield the viral RNA from detection by cytosolic innate immune sensors.
Lecture 21: RNA Synthesis in Eukaryotes Flashcards | …
To examine whether BrUTP is similarly incorporated into viral RNA in cells, we transfected 5 mM BrUTP into BSR-T7 cells that were infected 6 hours earlier with VSV. Infected cells were subsequently exposed to [3H]-uridine in the presence of ActD to permit the labeling of viral RNA, and the total cellular RNA was extracted, purified and BrUTP incorporation determined by immune precipitation prior to electrophoresis on acid-agarose gels. Consistent with the incorporation of BrUTP by the VSV polymerase in vitro, viral mRNAs were immune precipitated from cells that were transfected with BrUTP, but not from cells that lacked BrUTP (). This set of experiments demonstrates that VSV L incorporates 5-BrUTP into viral mRNA in vitro and in infected cells.
To visualize the cellular localization of viral RNA, we infected BSR-T7 cells with rVSV-RFP-P, and 5 hours later treated the cells with ActD to inhibit cellular transcription and glucosamine to deplete the intracellular pool of uridine , . Following a 1 hour incubation, the RNA was labeled by incorporation of BrUTP for 1 hour and was subsequently visualized by IF microscopy. In infected cells - as evidenced by the RFP-P inclusions - we found BrUTP labeled RNA distributed throughout the cytoplasm (, row 1, arrows). No BrUTP labeled RNA was detected in uninfected cells (, rows 1 and 2). As expected, in the absence of ActD we observed BrUTP labeled cellular RNA, which was predominantly localized to the nucleus (, row 3, arrowheads), and no RNA was visualized in cells that did not receive BrUTP (, row 4). This result shows that VSV RNA is localized throughout the cytoplasm in infected cells. We could not discriminate, however, whether viral RNA was synthesized throughout the cytoplasm, or at the RFP-P inclusions followed by subsequent movement. Consistent with this latter idea, we detected BrUTP labeled RNA in close proximity to inclusions as well as throughout the cytoplasm when BrUTP incorporation was allowed to proceed for only 30 minutes prior to fixation (, arrows).
10.3: Structure and Function of RNA - Biology LibreTexts
For immunogold EM, infected cells were fixed 6 hpi with 2% paraformaldehyde (Sigma) and labeled with primary antibodies against viral L (1100 dilution) and N (150 dilution) as above. To detect P, we infected cells instead with VSV-eGFP-P and visualized the location of P with a rabbit anti-GFP antibody (150 dilution) (Sigma). Secondary labeling was performed with anti-rabbit or anti-mouse nanogold-1.4 nm (150 dilution) in 1% BSA for 1 h at RT. Samples were washed 5× in 1× PBS/1% BSA for 1h and postfixed in 1% glutaraldehyde (Electron Microscopy Sciences) in 1× PBS for 10 min. Cells were then washed 3 times for 5 min in PBS, followed by 2 washes for 5 min in deionized water and 1 wash for 5 min in 0.02 M citrate buffer. The 1.4 nm gold particles were silver enhanced (giving ~15–40 nm particles) by incubating the samples for 4 min in freshly mixed developer using the HQ Silver Enhancement kit (Nanoprobes, Yaphank, NY) and rinsed 3 times in deionized water for 1 min. Cells were treated with 0.5% osmium tetroxide before dehydration.
Structure and Function of RNA ..
To confirm that viral RNA was transported away from inclusions, we performed a pulse-chase analysis. To do this, we first depleted intracellular pools of uridine with glucosamine (+Gluc) , , labeled the RNA by incorporation of BrUTP and then subsequently “chased” with a 10-fold excess of unlabeled uridine (see schematic in ). When nocodazole was absent during the indicated chase period, the viral RNA granules were found a range of distances away from the inclusions rather than closely surrounding them (). This observation confirmed that the RNA was transported away from the inclusions in a microtubule-dependent manner and suggests that this is an active process. Consistent with this notion, RNA granules were observed along and in close proximity to microtubules (, enlarged inset, arrows). These RNA localization experiments reveal that VSV RNA is synthesized at inclusions in infected cells and that the viral RNA is transported away from those inclusions in a microtubule-dependent manner to become distributed throughout the cytoplasm.
Structure and Function of RNA; ..
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.