Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos 62210, Mexico
Correspondence
Carlos F. Arias
arias{at}ibt.unam.mx
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published online ahead of print on 31 March 2005 as DOI 10.1099/vir.0.80827-0.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the process of infection, the infecting TLP is adsorbed, penetrates the plasma membrane and is uncoated, loosing the two outer layer proteins and yielding a transcriptionally active DLP. The viral polymerase VP1 then synthesizes the primary viral transcripts, which in addition to direct the synthesis of viral proteins, i.e. to function as mRNAs, are also believed to serve as RNA templates [RNA(+)] for the synthesis of the RNA-negative strands [RNA()] to form the genomic dsRNA (Estes, 2001; Patton et al., 2003
). Once a critical mass of viral proteins is synthesized, viral polypeptides NSP2, NSP5, NSP6, VP1, VP2, VP3 and VP6 accumulate in electron-dense cytoplasmic inclusions known as viroplasms (González et al., 2000
; Mattion et al., 1991
; Petrie et al., 1984
); these are key structures in the replication of rotavirus, where the synthesis of dsRNA and assembly of progeny DLPs are thought to take place (Estes, 2001
).
The synthesis of RNA() has been proposed to occur concurrently with the packaging of RNA(+) into core replication intermediate (RI) particles (formed by VP1, VP2, VP3, and the non-structural proteins NSP2 and NSP5) in a highly coordinated manner, such that packaging and replication of RNA(+) lead to the formation of cores containing one copy of each of the 11 dsRNA genome segments (Patton et al., 2003). Assembly of VP6 onto core RIs is then believed to lead to the production of transcriptionally active, dsRNA-containing DLPs (Estes, 2001
). These particles are thought to initiate a second round of transcription, which results in an amplified second wave of viral protein synthesis and assembly of DLPs, as has been shown for reovirus (Nibert & Schiff, 2001
). Finally, the assembled DLPs bud through the membrane of the ER, acquiring during this process a transient membrane envelope, which is subsequently removed to yield mature infectious TLPs (Estes, 2001
; Patton, 1995
). Although the mechanism through which the transient membrane envelope is lost is not known, it has been recently shown to depend on VP7 (López et al., 2005
).
Rotavirus gene 11 encodes two proteins, NSP5 and NSP6. NSP5 is an O-glycosylated phosphoprotein (Afrikanova et al., 1996; González & Burrone, 1991
) present in several phosphorylated isoforms, which are thought to regulate their own phosphorylation (Afrikanova et al., 1996
; Blackhall et al., 1998
; Eichwald et al., 2004
; Eichwald et al., 2002
). NSP5 has been shown to interact in a sequence independent fashion with dsRNA and single-stranded (ss) RNA (Vende et al., 2002
), and it has been suggested that its phosphorylation state influences the rate of translation versus replication of the viral RNA (Chnaiderman et al., 2002
). When co-expressed with NSP2, in the absence of other viral proteins, NSP5 forms viroplasm-like structures (Fabbretti et al., 1999
), and more recently it was shown that amino-terminal deletion mutants of this protein can form viroplasm-like structures in the absence of NSP2 (Mohan et al., 2003
). NSP5 has also been shown to have a strong affinity to VP2, to an extent that it can outcompete VP6 in VLPs (Berois et al., 2003
). Despite the extensive characterization of the biochemical properties of NSP5, and the description of its viroplasm-forming properties by transient expression in uninfected cells, the role and relevance of this protein in the context of virus replication has not been determined.
NSP6 is a 92 aa protein of unknown function, encoded in a +1 alternative open reading frame (ORF) of rotavirus gene 11 (Mattion et al., 1991). The NSP5 gene of many rotavirus strains has an ORF that could potentially encode NSP6; however, its synthesis has only been demonstrated in a few rotavirus strains (Mattion et al., 1991
; Torres-Vega et al., 2000
). In addition, the fact that some rotaviruses do not have this ORF suggests that NSP6 is not essential for virus replication (Mattion et al., 1991
; Taraporewala & Patton 2004
; Torres-Vega et al., 2000
).
RNA interference (RNAi) is a process triggered by dsRNA that specifically silences the expression of the gene which shares sequence identity with the triggering dsRNA. The recent implementation of the RNAi technology to efficiently silence gene expression in mammalian cells has opened a tremendous opportunity for a rapid advance in the characterization of gene function (Elbashir et al., 2001). This technology has also proven to be extremely useful to study the function of genes from plant and animal viruses, including rotaviruses (Arias et al., 2004
; Dector et al., 2002
; López et al., 2005
; Silvestri et al., 2004
).
To evaluate directly the role of NSP5 in the context of the replication of rotaviruses in infected cells, we silenced the expression of rotavirus RRV gene 11 by RNAi. We found that reduced amounts of NSP5 correlated with the inhibition of genomic dsRNA and viral protein synthesis, as well as with a marked reduction in the production of infectious viral progeny. These data indicate an essential role of NSP5 in the replication of rotaviruses, and give further insight into the function this protein plays in the virus replication cycle.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequence and lipofection of small interfering RNAs (siRNAs).
For this work two NSP5 siRNAs corresponding to nt 95115 (siRNANSP5-1) and 244264 (siRNANSP5-2) of rotavirus RRV gene 11 (Mohan & Atreya, 2001) were purchased from Dharmacon Research. siRNANSP5-1 had the sequence 5'-UCUAUUGGUAGGAGUGAACTT-3' (sense), 5'-GUUCACUCCUACCAAUAGATT-3' (antisense), while siRNANSP5-2 had the sequence 5'-GACAAAUGCAGACGCUGGCTT-3' (sense), 5'-GCCAGCGUCUGCAUUUGUCTT-3' (antisense). Both NSP5 siRNAs silenced the expression of gene 11 with similar efficiency. As a negative control a previously reported lamin siRNA was used (Dector et al., 2002
; Elbashir et al., 2001
). Confluent monolayers of MA104 cells in 48- or 96-well plates were lipofected with 100 or 50 µl, respectively, of a mixture containing 40 µg lipofectamine (Invitrogen) ml1 and 600 pmol siRNAs ml1 in MEM without serum. The transfection mixture was added to cells previously washed with MEM and incubated for 8 h at 37 °C. After this time the transfection mixture was removed, the cells were washed with MEM and kept in this medium without serum or antibiotics for 36 h at 37 °C, before being infected at an m.o.i. of 3.
Infection of cells and titration of viral progeny.
Cell monolayers in 96-well plates were infected with an m.o.i. of 3 as described previously (Pando et al., 2002), and then incubated for 1416 h at 37 °C. At this time the cells were lysed by two freezethaw cycles, and the lysates were treated with 10 µg trypsin ml1 for 30 min at 37 °C. The infectious titre of the viral preparations was obtained by an immunoperoxidase focus assay (Pando et al., 2002
).
Immunoblots.
Cells were transfected with siRNAs and infected with individual rotavirus strains RRV, Wa, RF or Alabama as described above. At 12 h post-infection (p.i.) the cells were lysed with Laemmli sample buffer, the proteins separated by 14 % SDS-PAGE and transferred to Immobilon-NC (Millipore). The transferred proteins were incubated with a mixture of rabbit polyclonal antibodies to purified rotavirus RRV, NSP5 and vimentin, and the bound antibodies were developed by incubation with a peroxidase-labelled anti-rabbit antibody (Perkin-Elmer Life Sciences) and the Western Lightning system (Perkin-Elmer).
Radiolabelling of proteins and RNA.
For protein labelling, cells grown in 48-well plates were transfected with siRNAs and infected with rotavirus RRV as described above. At the indicated times, the medium was replaced by MEM without methionine, supplemented with 50 µCi (1·85 MBq) Easy Tag Express 35S-labelling mix (Dupont NEN) ml1 and incubated for 30 min; after this period the cells were washed and lysed with Laemmli sample buffer. The relative concentration of protein in each sample was adjusted by eye inspection of Coomassie blue-stained gels, and the same amount of protein in each sample was resolved by 11 % SDS-PAGE. For RNA labelling, cells grown in 12-well plates were transfected with siRNAs and infected with rotavirus RRV as described above. After the virus adsorption period, actinomycin (5 µg ml1; Sigma) was added to the cells; 1 h later 50 µCi [32P]orthophosphate (Dupont NEN) ml1 was added in phosphate-free MEM containing actinomycin, and the cells were further incubated for 10 h at 37 °C. At this time total RNA was extracted from the cells with Trizol (Invitrogen). ssRNA and dsRNA were resolved in denaturing 7 M urea polyacrylamide gels as described previously (Silvestri et al., 2004). Radiolabelled RNAs were detected with a Molecular Dynamics PhosphorImager.
Analysis of genomic dsRNA.
Genomic dsRNA was isolated from viral lysates and analysed by PAGE and silver staining as described previously (Herring et al., 1982).
Immunofluorescence.
MA104 cells were grown on coverslips and infected as described above. The cells were fixed 8 h p.i. with 2 % paraformaldehyde and permeabilized with 0·5 % Triton X-100 in 1 % BSA, as described previously (Dector et al., 2002). The cells were then incubated with either rabbit polyclonal sera to NSP2, NSP5 or VP4, or with mAbs 3A8, 255/60, 60 or B4, followed by staining with goat anti-mouse IgG coupled to Alexa-568 or goat anti-rabbit IgG coupled to Alexa-488 (Molecular Probes). The slides were analysed with a Bio-Rad MRC-600 confocal microscope and CoMOS MPL-1000 software.
Real-time RT-PCR.
The level of gene 10 RNA(+), which includes both gene 10 mRNA and the positive-strand RNA present in gene 10 dsRNA, was quantified by real-time RT-PCR. Briefly, MA104 cells grown in 12-well plates were transfected with siRNAs and infected with rotavirus RRV as described above. At the indicated times the cells were lysed with Trizol and total RNA was purified according to manufacturer's instructions. cDNA was synthesized from 0·1 to 1 ng of total RNA using primer 5'-TCCTGGAATGGCGTATTTTC-3', which corresponds to nt 122141 of RRV gene 10. The DNA was then amplified in the presence of a SybrGreen PCR master mix (Applied Biosystems) by addition of reverse primer 5'-GAGCAATCTTCATGGTTGGAA-3' (nt 173193 of RRV gene 10), using an ABI Prism 7000 detection system (Applied Biosystems). The results were normalized to the levels of GAPDH mRNA detected in each RNA sample (Winer et al., 1999).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Interference of rotavirus gene 11 expression reduces the general synthesis of viral mRNAs and genomic dsRNA
To analyse the effect of silencing the expression of gene 11 on the synthesis of viral messenger and genomic RNA, siRNANSP5-1-transfected cells were infected with RRV, the cells were labelled with [32P]orthophosphate for 10 h, starting 2 h p.i., followed by the extraction of total RNA from the cell lysates and analysis by PAGE, using the conditions described by Silvestri et al. (2004) that allow the detection of viral mRNAs and most segments of dsRNA (Fig. 2a
). A reduction in the accumulation of all viral transcripts and dsRNA was observed in the presence of siRNANSP5-1 as compared with cells transfected with an irrelevant siRNA. The amount of viral dsRNA synthesized was also analysed by PAGE and silver staining of unlabelled dsRNA extracted at 12 h p.i. Similar to what was observed for 32P-labelled RNA, the amount of dsRNA was also found to be diminished in cells transfected with siRNANSP5-1, as compared with cells transfected with a control siRNA or with an siRNA to RRV VP4 (Dector et al., 2002
) (Fig. 2b
).
|
|
|
Silencing the expression of gene 11 blocks late viral transcription and protein synthesis
The reduced synthesis of viral protein, mRNA, dsRNA and progeny particles described above, suggests that the inhibition of gene 11 expression blocks the replication of the viral genome and the putative second round of transcription. If this is correct, one would expect that the primary synthesis of viral mRNA and protein that takes place early after infection would not be affected (except for NSP5), but the inhibition would be evident rather late in the replication cycle, when the secondary round of transcription and translation occurs. To test this hypothesis, MA104 cells were infected with rotavirus RRV at an m.o.i. of 3, and the infected cells were pulse-labelled every hour for 30 min with an 35S-labelling mix, starting 1 h after infection (Fig. 5a). The level of NSP5 was detected by immunoblot using the same samples shown in Fig. 5
(a) (Fig. 5b
). The inhibition of the synthesis of NSP5 was already evident at 2 h p.i., a time point at which this protein was first detected in control cells. In contrast, the synthesis of the rest of the viral proteins was similar in the NSP5 siRNA- and control siRNA-transfected cells until 3 h p.i., while the inhibition of their synthesis started to be noticed at 4 h p.i. and was maximal at 68 h p.i.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the RNAi response induced by siRNANSP5-1 most probably inhibits the synthesis of both NSP5 and NSP6, the block in virus replication, as well as the reduced synthesis of viral components observed in this work is most likely the result of the impaired levels of synthesis of NSP5. This conclusion is supported by the following observations: (i) an ORF encoding a full-length NSP6 is not present in gene 11 of some group A rotaviruses (strains Mc323, Alabama, 512-C and OSU GenBank accession numbers U54772, J04361, AB008662 and X15519, respectively) and is missing in all group C rotavirus strains (Torres-Vega et al., 2000), suggesting that NSP6 is not essential for the replication of the virus; (ii) the expression of intracellular antibodies to NSP5 caused a phenotype similar to that described in this work (Vascotto et al., 2004
); (iii) silencing the expression of gene 11 of porcine rotavirus strain OSU, which encodes a truncated NSP6 ORF, resulted in the general reduction of viral protein synthesis and inhibition of rotavirus replication (Campagna et al., 2003
.); and (iv) the inhibition effect of siRNANSP5-2 on the replication and protein synthesis of rotavirus Alabama described in this work was similar to that observed for rotavirus RRV. The gene 11 of Alabama strain does not have an AUG codon at the beginning of the ORF that would code for a 92 aa NSP6 protein, like most other rotavirus strains. It rather has an in-phase downstream AUG that could potentially code for a protein of 79 aa. If this protein is indeed synthesized and if it is functional, it would imply that the 79 carboxy-terminal amino acids of NSP6 are enough to sustain its function. Furthermore, as mentioned above, rotavirus OSU gene 11 produces a 51 aa truncated version of NSP6, and its silencing also causes a reduction of viral protein and genomic dsRNA synthesis (Campagna et al., 2003
). Although it is not possible to discard a central role of NSP6 in rotavirus replication, these observations suggest that most probably NSP6 plays a regulatory role of the NSP5 activity, as previously suggested (Torres-Vega et al., 2000
), a role that might have been substituted somehow in the strains that lack the full-length NSP6.
Gene 11-silenced cells showed fewer and smaller viroplasms than cells transfected with the control siRNA, as well as a partial delocalization of viroplasmic proteins VP2, NSP2 and VP6 (Fig. 4), strongly suggesting that NSP5 is essential to nucleate the formation of these cytoplasmic inclusions (Fabbretti et al., 1999
). It would appear that in the absence of a regular amount of NSP5 the number and size of viroplasms decreases, causing that only a fraction of viroplasmic proteins anchors to these structures, while the excess' of these proteins redistributes in the cytoplasm. In contrast to NSP2 and VP2, whose excess' became homogeneously distributed in the cytoplasm, VP6 showed a dramatic redistribution into fibrous structures that appear to extend to the periphery of the cell. The reason for this fibrous pattern of VP6 is not clear, since the VP6 fibres did not co-localize with vimentin, actin or tubulin, and had variable thickness (data not shown). The distribution of VP6 into fibres was also observed when the expression of NSP4 was silenced (López et al., 2005
); in that case, the amounts of NSP2 and NSP5 were also reduced, and the mean size of the viroplasms was also smaller, suggesting that the distribution of VP6 is sensitive to the concentration of proteins that accumulate in viroplasms and that treatments that alter this concentration cause VP6 to redistribute as fibres. The reason for this delocalization is not clear, but NSP5 might have an unrecognized role in the recruitment of viral proteins to viroplasms. Direct interaction with NSP5 has been demonstrated for NSP6, NSP2, VP1 and VP2, and changes in the distribution of VP2 were observed when NSP5 was co-expressed in insect cells (Berois et al., 2003
). In the case of VP6, there are no reports describing a direct interaction with NSP5 and, in fact, NSP5 has been reported to dislodge VP6 from virus-like particles formed by VP2 and VP6 (Berois et al., 2003
). Recently, it was shown that the inhibition of NSP2 expression also inhibits viroplasm formation, genome replication, virus assembly, and the synthesis of the viral proteins (Silvestri et al., 2004
); however, the intracellular distribution of the viral proteins in NSP2-deficient cells was not characterized. VP4, VP7 and NSP4, which are not integral viroplasmic proteins but are closely associated to these structures (González et al., 2000
), were also delocalized in gene 11-silenced cells, probably as an indirect effect of the reduced size and number of viroplasms.
The data presented in this work support the existence of a second round of transcription driven by de novo synthesized DLPs (Fig. 5). The impaired number and size of viroplasms produced in gene 11-silenced cells probably causes a decreased production of DLPs that results in a deficient secondary synthesis of viral mRNA, with the consequent reduction of: (i) the synthesis of viral proteins and genomic dsRNA; (ii) the formation of healthy viroplasms; and (iii) the assembly of DLPs, and eventually infectious TLPs. As predicted by this model, early after virus infection (3 h or earlier) of siRNANSP5-transfected cells only the accumulation of NSP5 (the siRNA target) is clearly affected (Fig. 5a
), while the synthesis of other viral proteins and of RNA(+), as judged from the levels of gene 10 RNA, are not affected, indicating that at the beginning of infection the RNA(+) is mostly produced by the parental infecting DLP. At later times (4 h p.i. and later) all viral proteins and viral RNA(+) start to be inefficiently produced because, in the absence of NSP5, viroplasms are scarce, and a limited amount of progeny DLPs are assembled, with the consequent reduction in viral protein and RNA(+), and an inhibition of the production of progeny infectious virus. Experiments are currently being carried out in our laboratory to characterize in detail the timing of transcription and replication of the virus genome during the rotavirus infection cycle, and the effect on this timing of silencing the expression of various structural and non-structural viral proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arias, C. F., Dector, M. A., Segovia, L., Lopez, T., Camacho, M., Isa, P., Espinosa, R. & Lopez, S. (2004). RNA silencing of rotavirus gene expression. Virus Res 102, 4351.[CrossRef][Medline]
Berois, M., Sapin, C., Erk, I., Poncet, D. & Cohen, J. (2003). Rotavirus nonstructural protein NSP5 interacts with major core protein VP2. J Virol 77, 17571763.
Blackhall, J., Muñoz, M., Fuentes, A. & Magnusson, G. (1998). Analysis of rotavirus nonstructural protein NSP5 phosphorylation. J Virol 72, 63986405.
Campagna, M., Vascotto, F., Eichwald, C. & Burrone, O. R. (2003). Interfering with rotavirus NSP5. In Eighth International Symposium on Double Stranded RNA Viruses. Italy: Castelvecchio Pascoli (Lucca).
Chnaiderman Xiao, J., Barro, M. & Spencer, E. (2002). NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells. Arch Virol 147, 18991911.[CrossRef][Medline]
Dector, M. A., Romero, P., López, S. & Arias, C. F. (2002). Rotavirus gene silencing by small interfering RNAs. EMBO Rep 3, 11751180.
Eichwald, C., Vascotto, F., Fabbretti, E. & Burrone, O. R. (2002). Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains. J Virol 76, 34613470.
Eichwald, C., Jacob, G., Muszynski, B., Allende, J. E. & Burrone, O. R. (2004). Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation. Proc Natl Acad Sci U S A 101, 1630416309.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001). Duplex of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498.[CrossRef][Medline]
Estes, M. K. (2001). Rotaviruses and their replication. In Fields Virology, 4th edn, pp. 17471785. Edited by D. N. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Fabbretti, E., Afrikanova, I., Vascotto, F. & Burrone, O. R. (1999). Two non-structural rotaviral proteins, NSP2 and NSP5, form viroplasm-like structures in vitro. J Gen Virol 80, 333339.[Abstract]
González, S. A. & Burrone, O. R. (1991). Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine. Virology 182, 816.[CrossRef][Medline]
González, R. A., Espinosa, R., Romero, P., López, S. & Arias, C. F. (2000). Relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells. Arch Virol 145, 19631973.[CrossRef][Medline]
Herring, A. J., Inglis, N. F., Ojeh, C. K., Snodgrass, D. R. & Menzies, J. D. (1982). Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels. J Clin Microbiol 16, 473477.[Medline]
Kim, D. H., Longo, M., Han, Y., Lundberg, P., Cantin, E. & Rossi, J. J. (2004). Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 22, 321325.[CrossRef][Medline]
López, T., Camacho, M., Zayas, M., Nájera, R., Sánchez, R., Arias, C. F. & López, S. (2005). Silencing the morphogenesis of rotavirus. J Virol 79, 184192.
Mattion, N. M., Mitchell, D. B., Both, G. W. & Estes, M. K. (1991). Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11. Virology 181, 295304.[CrossRef][Medline]
Mohan, K. V. K. & Atreya, C. D. (2001). Nucleotide sequence analysis of rotavirus gene 11 from two tissue culture-adapted ATCC strains, RRV and Wa. Virus Genes 23, 321329.[CrossRef][Medline]
Mohan, K. V. K., Muller, J., Som, I. & Atreya, C. D. (2003). The N- and C-terminal regions of rotavirus NSP5 are critical determinants for the formation of viroplasm-like structures independent of NSP2. J Virol 77, 1218412192.
Nejmeddine, M., Trugnan, G., Sapin, C., Kohli, E., Svensson, L., López, S. & Cohen, J. (2000). Rotavirus spike protein VP4 is present at the plasma membrane and is associated with microtubules in infected cells. J Virol 74, 33133320.
Nibert, M. L. & Schiff, L. A. (2001). Reovirus and their replication. In Fields Virology, 4th edn, pp. 16791728. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Pando, V., Isa, P., Arias, C. F. & Lopez, S. (2002). Influence of calcium on the early steps of rotavirus infection. Virology 295, 190200.[CrossRef][Medline]
Patton, J. T. (1995). Structure and function of the rotavirus RNA-binding proteins. J Gen Virol 76, 26332644.[Medline]
Patton, J. T., Kearny, K. & Taraporewala, Z. F. (2003). Rotavirus genome replication: role of the RNA-binding proteins. In Viral Gastroenteritis, pp. 165184. Edited by U. Desselberger & J. Gray. Amsterdam: Elsevier.
Persengiev, S. P., Zhu, X. & Green, M. R. (2004). Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 1218.
Petrie, B. L., Greenberg, H. B., Graham, D. Y. & Estes, M. K. (1984). Ultrastructural localization of rotavirus antigens using coloidal gold. Virus Res 1, 133152.[CrossRef][Medline]
Silvestri, L. S., Taraporewala, Z. F. & Patton, J. T. (2004). Rotavirus replication plus-sense templates for double-stranded RNA synthesis are made in viroplasms. J Virol 78, 77637774.
Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H. & Williams, B. R. (2003). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834839.[CrossRef][Medline]
Taraporewala, Z. F. & Patton, J. T. (2004). Nonstructural proteins involved in genome packaging and replication of rotaviruses and others members of the Reoviridae. Virus Res 101, 5766.[CrossRef][Medline]
Torres-Vega, M. A., González, R. A., Duarte, M., Poncet, D., López, S. & Arias, C. F. (2000). The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6. J Gen Virol 81, 821830.
Vascotto, F., Campagna, M., Visintin, M., Cattaneo, A. & Burrone, O. R. (2004). Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle. J Gen Virol 85, 32853290.
Vende, P., Taraporewala, Z. F. & Patton, J. T. (2002). RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA. J Virol 76, 52915299.
Winer, J., Jung, C. K. S., Shackel, I. & Williams, M. (1999). Development and validation of a real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270, 4149.[CrossRef][Medline]
Zarate, S., Cuadras, M. A., Espinosa, R., Romero, P., Juarez, K. O., Camacho-Nuez, M., Arias, C. F. & Lopez, S. (2003). Interaction of rotaviruses with Hsc70 during cell entry is mediated by VP5. J Virol 77, 72547260.
Received 15 December 2004;
accepted 20 March 2005.