Rotavirus genome replication: Some assembly required

Long, Courtney P.; McDonald, Sarah

doi:10.1371/journal.ppat.1006242

Cited by 32 publications

(35 citation statements)

References 23 publications

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“…Specifically, transcription of ϩRNAs occurs within DLPs, while replication of the dsRNA genome occurs within assembly intermediates. VP2 is critical for VP1-mediated dsRNA synthesis in vitro and in vivo; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood (7)(8)(9)(14)(15)(16)(17)(18)(19)(20). Unfortunately, no structural information exists for the assembly intermediates, so the mode of VP1-VP2 interaction during dsRNA is not known.…”

Section: Discussionmentioning

confidence: 99%

“…However, the role of the core shell protein goes beyond merely serving as a physical framework upon which the polymerase functions. The capacity of VP1 to synthesize RNA, in vitro and in vivo, is dependent upon the presence of VP2, suggesting that the core shell protein is also a critical enzymatic activator of the polymerase (7)(8)(9)(14)(15)(16)(17)(18)(19)(20). However, the precise contact sites between VP1 and VP2 that culminate in polymerase activation and particleassociated RNA synthesis are poorly understood.…”

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confidence: 99%

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In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

Steger

Brown

Sullivan

et al. 2019

J Virol

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The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-fold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation. IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

Steger

Brown

Sullivan

et al. 2019

J Virol

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“…Rotavirus strain ZTR-68 cultured in MA104 cells named M-ZTR-68 and that cultured in Vero cells named V-ZTR-68. Simian rotavirus strain SA11 (G3P [2]), human rotavirus strain S2 (G2P [4]) and porcine rotavirus strain Gottfried (G4P [8]) were kept in our lab. The wild rotavirus strain ZTR-18 (G9P [8]) was isolated from a fecal sample from a infant in Yunnan province, China in 2013.…”

Section: Cells and Virusmentioning

confidence: 99%

“…Following infection, loss of the VP4 and VP7 outer layers yields the transcriptionally active DLP, activating gene transcription and viral protein synthesis. Replication of the rotavirus genome and assembly of the DLPs are primarily accomplished within the viroplasm, which is formed by the non-structural viral proteins NSP2 and NSP5 [4].…”

Section: Introductionmentioning

confidence: 99%

MicroRNA-7 Inhibits Rotavirus Replication by Targeting Viral NSP5 In Vivo and In Vitro

Zhou

Chen

et al. 2020

Viruses

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Rotavirus (RV) is the major causes of severe diarrhea in infants and young children under five years of age. There are no effective drugs for the treatment of rotavirus in addition to preventive live attenuated vaccine. Recent evidence demonstrates that microRNAs (miRNAs) can affect RNA virus replication. However, the antiviral effect of miRNAs during rotavirus replication are largely unknown. Here, we determined that miR-7 is upregulated during RV replication and that it targets the RV NSP5 (Nonstructural protein 5). Results suggested that miR-7 affected viroplasm formation and inhibited RV replication by down-regulating RV NSP5 expression. Up-regulation of miR-7 expression is a common regulation method of different G-type RV-infected host cells. Then, we further revealed the antiviral effect of miR-7 in diarrhea suckling mice model. MiR-7 is able to inhibit rotavirus replication in vitro and in vivo. These data provide that understanding the role of cellular miR-7 during rotaviral replication may help in the identification of novel therapeutic small RNA molecule drug for anti-rotavirus.

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“…РВИ эффективно инициируется, когда DLP попадает в цитоплазму и начинается синтез вирусных транскриптов. Внешний слой, так же как и средний, является икосаэдрическим капсидом и состоит из 260 тримеров гликопротеина VP7 (Т = 13) и содержит 60 шипов, образованных асимметричными тримерами белка VP4 [9][10][11][12][13][14]. Белки VP4 и VP7 являются главными мишенями вируснейтрализующих антител.…”

Section: структура вириона ротавирусовunclassified

Rotavirus Vaccines: New Strategies and Approaches

Кондакова

Nikitin

Trifonova

et al. 2017

Moscow Univ. Biol.Sci. Bull.

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Rotavirus infection is a rotavirus-associated disease. It is the main cause of severe diarrhea among children all over the world and one of the factors influencing the infant mortality. Today, only live attenuated vaccines are widely used for vaccination against rotavirus infection. Existing vaccines have demonstrated their effectiveness, but they have a number of side effects, primarily the risk of intussusception. Complications associated with the use of existing vaccines are usually associated with oral administration of drugs and the replication of attenuated live vaccines in the human intestine. In this regard, there is a need to design modern, effective and safe vaccines against rotavirus infection, unable to reproduce (to replicate) in the human body. Currently, modern vaccines against rotavirus infection are being developed and tested actively. These are recombinant vaccines with parenteral administration. The complicated antigenic structure of rotavirus is one of the main problems for the design of such recombinant vaccines. This review discusses the genetic and antigenic diversity of rotavirus strains and the geographical location of their epidemically significant variants. The role of capsid proteins in the formation of an immune response to the virus and the current state of development of new candidate recombinant vaccines against rotavirus infection are considered.

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Rotavirus genome replication: Some assembly required

Cited by 32 publications

References 23 publications

In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

MicroRNA-7 Inhibits Rotavirus Replication by Targeting Viral NSP5 In Vivo and In Vitro

Rotavirus Vaccines: New Strategies and Approaches

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