Acting Chief, Rotavirus Molecular Biology Section, LID
Our research is focused on the study of rotaviruses (RVs), which are the primary cause of severe dehydrating diarrhea in infants and young children. Globally, RV infections result in more than 500,000 deaths each year, with the vast majority occurring in developing countries. Despite their pathogenic importance, many of the fundamental events in the replication cycle of these segmented double-stranded RNA (dsRNA) viruses are not understood. A primary mission of our work is to acquire knowledge about the molecular biology, immunology, and epidemiology of RVs that can be applied to developing methods for preventing RV-induced disease.
RVs are non-enveloped, icosahedral, triple-layered particles (TLPs) that encapsidate genomes of 11 segments of dsRNA (Panel A and B). The outer layer of the TLP consists of the glycoprotein VP7 (yellow in Panel B) and the spike protein VP4 (red), while the intermediate layer is built from VP6 (blue). The inner protein layer is formed from 12 decamers of the core lattice protein VP2 (green). Positioned at the underside of the core vertices are complexes of the viral RNA-dependent RNA polymerase VP1 (ribbon structure in panel D) and the RNA capping enzyme VP3. Each dsRNA genome segment is associated with a single VP1-VP3 enzyme complex within the core.
Rotavirus entry into the cell is accompanied by the loss of the outer VP4-VP7 layer, thereby converting the TLP into a double-layered particle (DLP) (Panel A). The VP1-VP3 enzyme complex of the DLP directs the synthesis of 11 species of capped, plus-sense RNA (+RNAs), which are extruded through channels located at the vertices of the particle. Translation of the +RNAs gives rise to six structural proteins and five-six nonstructural proteins. The +RNAs also serve as templates for the synthesis of dsRNA genome segments. RNA replication occurs concurrently with packaging of the +RNAs into newly formed cores and is coordinated such that the 11 genome segments are produced at equimolar levels.
RNA replication and core formation take place in perinuclear, non-membrane-bound cytoplasmic inclusions (viroplasms) (green ringed structures in panel C). The building blocks of the viroplasm are the NSP2 octamer (ribbon structure in panel D) and the phosphoprotein NSP5. At the periphery of the viroplasm, progeny cores interact with VP6, yielding DLPs. These particles are recruited to the endoplasmic reticulum (ER) via affinity for the viral ER-transmembrane glycoprotein NSP4. NSP4 forms complexes with VP7 in the ER membrane, creating target regions through which DLPs bud into the ER lumen. The budding process culminates in the assembly of the VP4-VP7 outer capsid layer of the virion.
RV genome packaging and replicationRV genome replication (dsRNA synthesis) and transcription (+RNA synthesis) are catalyzed by VP1 within the confines of the virus core. VP1 is a globular protein composed of N-terminal, polymerase, and C-terminal domains (Panel E). The catalytic region is located in the hollow center, with four tunnels that connect to the surface VP1. The tunnels permit (i) entry of nucleotides, (ii) entry of template RNA, (iii) egress of dsRNA product or negative-sense RNA template, and (iv) egress of +RNA. Amino acid residues lining the RNA entry tunnel allow VP1 to specifically recognize the highly conserved 3' terminus of RV +RNAs. Although VP1 alone can form complexes with +RNAs, the protein can only synthesize dsRNA from +RNAs when the core lattice protein VP2 is present. The VP2-dependent nature of VP1 polymerase activity ensures that genome replication only occurs when sufficient VP2 is present to package newly made dsRNAs.
We hypothesize that VP2 induces two changes in VP1 that lead to polymerase activation. First, VP2 may bring about a re-positioning of 3’ end of +RNA in the catalytic region such that it is appropriately aligned with incoming nucleotides. And second, VP2 may trigger a conformation change in the priming loop, a flexible element of VP1 that stabilizes a nucleotide for RNA initiation in the catalytic region. In our laboratory, we are continuing studies designed to identify residues and structural elements critical to the function of the RNA polymerase, including defining VP1-VP2 interactive sites.
Secretion of Type I interferon by virus-infected cells is essential for turning on pathways that promote cellular transition to an antiviral state. In most mammalian cells, IFN production is initiated by activation of the constitutively expressed IFN regulatory factor (IRF)3, which in turn leads to induction of IRF7, the ”master regulator” of IFN synthesis. Early studies established that RV growth is impeded by IFN, and that the virus can circumvent this effect in naïve cells through a mechanism that subverts IFN expression. More recently, we and others found that RVs inhibit IFN signaling by inducing the degradation of not only IRF3, but also IRF7, with both turnover events occurring through proteasome-dependent processes.
The capacity of NSP1 to induce IRF7 degradation may allow RV to replicate in specialized trafficking cells (dendritic cells and macrophages), enabling the virus to move across the gut barrier and cause viremia. Along with IRF3 and IRF7, NSP1 was found to induce the degradation of IRF5, a factor that upregulates IFN expression and that is involved in triggering apoptosis. These results suggest that NSP1 functions as a broad-spectrum antagonist of IRF function.
Polyubiquitination places signals on a protein that promote its recognition and degradation by proteasomes. The fact that NSP1 mediates IRF3, IRF5, and IRF7 degradation through a proteasome-dependent process indicates that the interaction of NSP1 can trigger the ubiquitination of IRF proteins. Ubiquitination involves a catalytic cascade of E1, E2, and E3 ligases acting together to transfer ubiquitin onto a target protein. A common feature of many E3 ligases is the presence of a RING domain or other closely related zinc-binding domain. Sequencing of more than 100 RV strains indicates that the N-terminal region of NSP1 protein contains a highly conserved RING-like domain. This motif, combined with its proteasome-dependent activity, suggests that NSP1 has an E3 ligase activity. Consistent with this hypothesis is the observation that mutation of conserved residues of the NSP1 RING domain interferes with the capacity of NSP1 to degrade IRF proteins. Ongoing studies in our laboratory are aimed at elucidating the structure of NSP1 and further defining its mechanism of action.
Despite the endemic nature of RVs and their importance as human pathogens, information regarding the genome sequences of these viruses is severely limited. The scarcity of such genomics data prevents a comprehensive molecular analysis of RV diversity and evolution and, moreover, precludes the application of rational molecular-based approaches to developing more effective RV vaccines through reverse genetics. To help close this gap in information, we initiated projects that have so far provided the complete genome sequences of over 100 RV strains and isolates. The viruses chosen for analyses include prototypic laboratory strains used for studies of RV biology, strains developed for use in animal model systems and as vaccine candidates, human isolates developed as serotype reference strains, and isolates recovered from stool material of hospitalized RV-infected children. Through a collaboration with the J. Craig Venter Institute, we have developed a high-throughput robotics pipeline to sequence numerous primary RV isolates. Our sequencing results to date suggest that gene reassortment among co-circulating RVs is balanced by pressures to maintain specific genome constellations (gene sets). We look forward to better understanding how such constellations reflect the co-evolution of viral proteins that interact during RV replication.
Reassortment, the exchange of gene segments between viruses, has been used extensively to study RV biology and to generate antigenically diverse vaccine candidates. However, the process of creating and isolating reassortant RVs is slow and time consuming and not always feasible. Conversely, the development of a cDNA-based reverse genetics system would allow the efficient manipulation of the RV genome by directed replacement and engineering of gene segments. Toward that goal, we have developed a novel reverse genetics method that allows us to produce single-gene recombinant RVs. This method is helper-virus-dependent and is aided by RNAi-targeting of the gene to be replaced. We have been successful in repeatedly isolating virus-containing recombinant-derived RNAs within a single round of virus selection, indicating that this method is efficient and reproducible. We intend to use this important new technology for developing new vaccine candidates, mapping and modifying antigenic epitopes, and probing the function of viral proteins in virus growth and virulence.
Trask SD, McDonald SM, Patton JT. Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol. 2012 Jan 23;10(3):165-77.
McDonald SM, Patton JT. Rotavirus VP2 core shell regions critical for viral polymerase activation. J Virol. 2011 Apr;85(7):3095-105.
Ogden KM, Ramanathan HN, Patton JT. Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication. J Virol. 2011 Mar;85(5):1958-69.
Arnold MM, Patton JT. Diversity of interferon antagonist activities mediated by NSP1 proteins of different rotavirus strains. J Virol. 2011 Mar;85(5):1970-9.
Trask SD, Taraporewala ZF, Boehme KW, Dermody TS, Patton JT. Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18652-7.
McDonald SM, Tao YJ, Patton JT. The ins and outs of four-tunneled Reoviridae RNA-dependent RNA polymerases. Curr Opin Struct Biol. 2009 Dec;19(6):775-82.
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Last Updated June 08, 2015