RNA silencing of rotavirus gene expression

Arias, Carlos F.; Déctor, Miguel Angel; Segovia, Lorenzo; López, Tomás; Camacho, Minerva; Iša, Pavel; Espinosa, Rafaela

doi:10.1016/j.virusres.2004.01.014

Cited by 35 publications

(29 citation statements)

References 61 publications

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“…In this work, we have taken advantage of the siRNA technology to evaluate the role of NSP4 and other viral proteins (VP7 and VP4) in the changes in Ca 2ϩ homeostasis occurring in infected cells. These techniques have successfully been used to study rotavirus biology (1,3,5,7,12,21,22,25,35,36 (13). The activation of Ca 2ϩ permeability induced by infection was also reduced by the silencing of VP7; however, it was reduced to a lesser extent.…”

Section: Discussionmentioning

confidence: 99%

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells

Zambrano¹,

Díaz

Peña

et al. 2008

J Virol

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Rotavirus infection of cells in culture induces major changes in Ca2؉ homeostasis. These changes include increases in plasma membrane Ca 2؉ permeability, cytosolic Ca 2؉ concentration, and total cell Ca 2؉ content and a reduction in the amount of Ca 2؉ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca 2؉ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca 2؉ permeability of the plasma membrane and cytosolic Ca 2؉ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca 2؉ permeability and Ca 2؉ pools released by agonist (ATP). In addition, the increase of total Ca 2؉ content (as measured by 45 Ca 2؉ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca 2؉ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca 2؉ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects. Viral-associated diarrhea remains one of the most common causes of morbidity and mortality among infants and young children. Worldwide estimations indicate that rotaviruses are the leading viral agent associated with severe diarrhea in children younger than 5 years old (20). In addition, rotavirus infections are also a main cause of diarrhea in calves, piglets, and the young of other animals of economic importance (20). Thus, further knowledge of the virus-cell interactions and the events leading to pathogenesis are necessary to improve or develop new strategies that may prevent or reduce the health and economic impact caused by rotavirus infections.Rotaviruses are members of the Reoviridae family. The rotavirus virion is icosahedral, nonenveloped, and composed of three concentric layers of proteins and a genome of 11 segments of double-stranded RNA. Each genomic segment, with the exception of segment 11, encodes one viral protein for a total of six structural (VP1 to VP7) and six nonstructural proteins (NSP1 to NSP6). The inner layer of the virion is formed by VP2 and also contains the RNA-dependent RNA polymerase VP1 and the guanylyltransferase/methylase VP3. The middle capsid is composed of the major virion...

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Section: Discussionmentioning

confidence: 99%

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells

Zambrano¹,

Díaz

Peña

et al. 2008

J Virol

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“…The modification in the cytosolic accumulation of some viral proteins correlated with the presence of smaller and fewer viroplasms in siRNA NSP4 -transfected cells compared to control cells, which may in turn cause a decrement of de novo assembled, transcriptionally active, double-layered replicative intermediate particles. This would prevent the second wave of transcription and translation that is thought to occur during the virus infectious cycle, with the consequent reduction in the final assembly of both DLPs and TLPs (1).…”

Section: Discussionmentioning

confidence: 99%

“…The produced mature virus is then released either by cell lysis in MA104 cells (13) or after transport along an atypical trafficking pathway from the ER to the plasma membrane in polarized epithelial Caco-2 cells (18). It has recently been shown that the expression of rotavirus genes can be efficiently silenced by RNA interference using small interfering RNAs (siRNAs) that have a sequence complementary to the viral gene to be silenced (1,9,32). siRNA has proven to be a very useful tool to dissect the function of the viral genes in the context of a natural infection (1,20).…”

mentioning

confidence: 99%

“…It has recently been shown that the expression of rotavirus genes can be efficiently silenced by RNA interference using small interfering RNAs (siRNAs) that have a sequence complementary to the viral gene to be silenced (1,9,32). siRNA has proven to be a very useful tool to dissect the function of the viral genes in the context of a natural infection (1,20). Silencing the expression of the rhesus rotavirus VP4 gene showed that in the absence of VP4, the DLPs were still able to bud into the ER and to lose the lipid envelope, yielding TLPs that lack VP4 spikes.…”

mentioning

confidence: 99%

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Silencing the Morphogenesis of Rotavirus

López

Camacho

Zayas

et al. 2005

J Virol

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113

118

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The morphogenesis of rotaviruses follows a unique pathway in which immature double-layered particles (DLPs) assembled in the cytoplasm bud across the membrane of the endoplasmic reticulum (ER), acquiring during this process a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (TLPs). In this work, we have characterized the role of NSP4 and VP7 in rotavirus morphogenesis by silencing the expression of both glycoproteins through RNA interference. Silencing the expression of either NSP4 or VP7 reduced the yield of viral progeny by 75 to 80%, although the underlying mechanism of this reduction was different in each case. Blocking the synthesis of NSP4 affected the intracellular accumulation and the cellular distribution of several viral proteins, and little or no virus particles (neither DLPs nor TLPs) were assembled. VP7 silencing, in contrast, did not affect the expression or distribution of other viral proteins, but in its absence, enveloped particles accumulated within the lumen of the ER, and no mature infectious virus was produced. Altogether, these results indicate that during a viral infection, NSP4 serves as a receptor for DLPs on the ER membrane and drives the budding of these particles into the ER lumen, while VP7 is required for removing the lipid envelope during the final step of virus morphogenesis.Rotaviruses are nonenveloped icosahedral viruses whose capsid is formed by three concentric layers of protein. The innermost layer is formed by 60 dimers of VP2 that surrounds the viral genome composed of 11 segments of double-stranded RNA and 12 copies of each VP1, the virus polymerase, and VP3, the virus capping enzyme. The second layer of protein is formed by 280 trimers of VP6, which sits on top of VP2 to form double-layered particles (DLPs). Finally, the addition of 280 trimers of glycoprotein VP7 which constitute the outermost layer of the virus and 60 dimeric spikes of the VP4 protein to DLPs form triple-layered particles (TLPs) that represent the mature infectious virus (13).Rotavirus morphogenesis occurs by an unusual process where DLPs, which are thought to assemble in cytoplasmic inclusions termed viroplasms, bud across the membrane of the endoplasmic reticulum (ER). During this process, the DLPs acquire a transient lipid envelope which is subsequently lost to yield the mature infectious TLPs (33). The ER membrane through which DLPs bud is modified by two viral proteins, the virion surface protein VP7 and the nonstructural polypeptide NSP4 (5, 7, 21). NSP4 has a large cytosolic domain that interacts with DLPs, and it has been proposed that this interaction drives the translocation of the double-layered particles into the lume...

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“…Hence, there is a need for alternative ways to treat this persistent infection. RNA interference (RNAi) has rapidly emerged as a technology for regulating mammalian gene expression because it provides a means of sequence-directed degradation of specific RNAs (1)(2)(3)(4)(5)(6)(7)(8). In the case of HBV, published in vivo hydrodynamic transfection studies have shown that simultaneous delivery of HBV expression plasmids and HBV-specific small interfering RNAs (siRNAs) (or siRNA-expressing constructs) to the mouse liver can prevent the induction of HBV gene expression and replication (9)(10)(11).…”

mentioning

confidence: 99%

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

Uprichard

Boyd

Althage

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

185

135

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Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBVspecific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.adenovirus vector ͉ RNA interference C hronic hepatitis B virus (HBV) infection causes Ͼ1 million deaths each year due to cirrhosis of the liver and hepatocellular carcinoma (www.cdc.gov͞hepatitis and www.who.int͞ mediacentre͞factsheets͞fs204͞en). Hence, there is a need for alternative ways to treat this persistent infection. RNA interference (RNAi) has rapidly emerged as a technology for regulating mammalian gene expression because it provides a means of sequence-directed degradation of specific RNAs (1-8). In the case of HBV, published in vivo hydrodynamic transfection studies have shown that simultaneous delivery of HBV expression plasmids and HBV-specific small interfering RNAs (siRNAs) (or siRNA-expressing constructs) to the mouse liver can prevent the induction of HBV gene expression and replication (9-11). To expand on those studies, we have examined the therapeutic potential of RNAi for the treatment of chronic HBV infection where ongoing viral gene expression and replication are established in the liver before siRNA delivery.Several variables that have not been previously addressed could affect the utility of RNAi for the treatment of an ongoing HBV infection. First, in the context of an established infection, viral RNAs may be protected within complexes or nucleocapsid structures as has been reported for hepatitis D virus (1), Rous sarcoma virus (12), and respiratory syncytial virus (RSV) (13). This is particularly relevant to HBV, which replicates in capsids after encapsidation of the viral pregenomic RNA. A second variable is how efficiently preexisting viral RNA can be reduced and to what extent RNA suppression can block viral DNA replication. Finally, established viral gene expression may allow for the induction of viral RNAi-defense mechanisms as reported for plant viruses (14) and flockhouse nodavirus (15).It has been shown that RNAi is capable of reducing mammalian gene expression in vivo. Genes such as fas (16), caspase-8 (17), agouti-related peptide (18), tyrosine hydroxylase (19), and ras (20, 21) have been targeted for degradation in mice, and corresponding changes in phenotype were observed. Yet, the actual degree o...

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RNA silencing of rotavirus gene expression

Cited by 35 publications

References 61 publications

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells

Silencing the Morphogenesis of Rotavirus

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

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RNA silencing of rotavirus gene expression

Cited by 35 publications

References 61 publications

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca 2+ Homeostasis Induced by Infection of Cultured Cells

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca 2+ Homeostasis Induced by Infection of Cultured Cells

Silencing the Morphogenesis of Rotavirus

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

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Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca ²⁺ Homeostasis Induced by Infection of Cultured Cells