Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms - PubMed (original) (raw)
Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms
Lynn S Silvestri et al. J Virol. 2004 Jul.
Abstract
Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.
Figures
FIG. 1.
Viral protein expression in siRNA-transfected cells. MA104 cells were transfected with the indicated siRNA, infected with SA11-5N (A and C) or DxRRV (B), and maintained in 35S-labeled amino acids. Lysates prepared from the cells at 18 h p.i. were analyzed for viral proteins by gel electrophoresis and autoradiography (lower panels) and by Western blot assay (upper panels). The specificity of the antibodies used in Western blot assays is given. Lysates prepared from cells neither transfected with siRNA nor infected with virus (uninfected) or infected with virus but not transfected with siRNA (no siRNA), were also analyzed.
FIG. 2.
IF analysis of the effect of siRNAs on intracellular accumulation of viral proteins. MA104 cells were transfected with the appropriate siRNA and infected with SA11-5N (A and C) or DxRRV (B). At 12 h p.i., the cells were processed for IF by using the indicated primary antibodies (α) and Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Cell nuclei were visualized by DAPI staining. For comparison purposes, panel C includes a cell that was not infected (arrowhead).
FIG. 3.
Level of viral dsRNAs produced in siRNA-transfected cells. MA104 cells were transfected with the indicated siRNA and infected with SA11-5N (A and C) or DxRRV (B). Cells were harvested at 18 h p.i. and analyzed for the presence of viral dsRNAs by PAGE and silver staining. The positions of the 11 genome segments are labeled.
FIG. 4.
Level of viral plus-strand RNAs produced in siRNA-transfected cells. MA104 cells were transfected with the indicated siRNA and infected with SA11-5N or DxRRV. (A) After cells were maintained in [32P]orthophosphate from 3 to 9 h p.i., RNAs were recovered and analyzed by electrophoresis on a urea-polyacrylamide gel and by autoradiography. The positions of viral plus-strand RNAs (1 to 11) and dsRNAs (ds1 to ds9) are shown. (B and C) Unlabeled RNAs were recovered from cells at 6 h p.i., resolved by electrophoresis, and transferred to nylon membranes. Blots were probed with 32P-labeled RNAs specific for SA11 g5 (SA11-g5PR) or g8 (SA11-g8PR) or for DxRRV g4 (RRV-g4PR) or g9 (D-g9PR), and the intensity of the bands was determined with a phosphorimager. The levels of the g9 and g5 plus- strand RNAs were calculated by dividing their band intensities by the corresponding intensities determined for g4 or g8 plus-strand RNAs, respectively. The values were then normalized, with the value for the IR samples taken as 100%.
FIG. 5.
Effect of siRNAs on virus titers. Lysates from MA104 cells infected with rotavirus and transfected with the indicated siRNA were analyzed for virus titer by plaque assay. Titers obtained from three separate assays performed in duplicate were averaged (values in parentheses [with standard errors]). Values were normalized to 100% for lysates from cells transfected with IR siRNAs.
FIG. 6.
Impact of g9-specific siRNA on virion assembly. MA104 cells were mock transfected or transfected with the g9D or IR siRNA, infected with DxRRV, and then maintained in 35S-labeled amino acids. DLPs and TLPs produced in the cells were resolved by CsCl centrifugation. (A) Bands of virus particles in the gradients were visualized with an inverted light source. (B) Fractions (three drops each) collected from the gradients in regions containing the bands were analyzed for density and for radioactivity in 10-μl aliquots. (C) Fractions containing DLPs (two to four in panel B) and TLPs (six to eight) were pooled and examined for protein content by PAGE and autoradiography.
FIG. 7.
Intracellular sites of RNA synthesis in rotavirus-infected cells. MA104 cells were infected with SA11-5N, treated with actinomycin D, and maintained in the absence of BrUTP (A) or in the presence of BrUTP from 8.5 to 9 h p.i. (B). Alternatively, the infected cells were maintained in actinomycin D from 8.5 to 9 h p.i. and then semifixed with paraformaldehyde, permeabilized with Triton X-100, and incubated in transcription buffer containing BrUTP (C). Subsequently, the cells were processed for IF analysis by using NSP2- and BrdU-specific antibodies.
FIG. 8.
Resistance of newly made viral dsRNA to degradation by dsRNA-specific RNase. MA104 cells were infected with SA11-5N or mock infected, treated with actinomycin D, and maintained in the presence of [32P]orthophosphate from 8.5 to 9 h p.i. Cytoplasmic extracts prepared from infected cells were incubated in the absence (lane 1) or presence (lane 2) of RNase V1 or deproteinized by phenol-chloroform extraction prior to incubation with RNase V1 (lane 3). An extract from mock-infected cells was incubated in the absence of RNase V1 (lane 4).
FIG. 9.
Fate of viral RNA transfected into rotavirus-infected cells. MA104 cells were infected with SA11-5N and, at 1 h p.i., transfected with BrU-labeled g9 plus-strand RNA. Cells were processed at 9 h p.i. for IF analysis with NSP2- and BrdU-specific antibodies.
References
- Altenburg, B. C., D. Y. Graham, and M. K. Estes. 1980. Ultrastructural study of rotavirus replication in cultured cells. J. Gen. Virol. 46:75-85. - PubMed
- Chasey, D. 1977. Different particle types in tissue culture and intestinal epithelium infected with rotavirus. J. Gen. Virol. 37:443-451.
- Chnaiderman, J., M. Barro, and E. Spencer. 2002. NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus-infected cells. Arch. Virol. 147:1899-1911. - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources