Biomedical subjects

Rodrigo Vasquez-Del Carpio

Publications and source records attributed to Rodrigo Vasquez-Del Carpio.

6 recordsLinked to original sources

PubMed2007

Coupling of rotavirus genome replication and capsid assembly.

The Reoviridae family represents a diverse collection of viruses with segmented double-stranded (ds)RNA genomes, including some that are significant causes of disease in humans, livestock, and plants. The genome segments of these viruses are never detected free in the infected cell but are transcribed and replicated within viral cores by RNA-dependent RNA polymerase (RdRP). Insight into the replication mechanism has been provided from studies on Rotavirus, a member of the Reoviridae whose RdRP can specifically recognize viral plus (+) strand RNAs and catalyze their replication to dsRNAs in vitro. These analyses have revealed that although the rotavirus RdRP can interact with recognition signals in (+) strand RNAs in the absence of other proteins, the conversion of this complex to one that can support initiation of dsRNA synthesis requires the presence and partial assembly of the core capsid protein. By this mechanism, the viral polymerase can carry out dsRNA synthesis only when capsid protein is available to package its newly made product. By preventing the accumulation of naked dsRNA within the cell, the virus avoids triggering dsRNA-dependent interferon signaling pathways that can induce expression and activation of antiviral host proteins.

Amino Acid Sequence↗

PubMed2006

Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities.

Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the gamma-beta phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5'-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its gamma-beta phosphoanhydride bond. Our results show that NSP2 hydrolyzes the gammaP from RNAs and NTPs through Mg(2+)-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5'-terminal gammaP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during gammaP hydrolysis.

Acid Anhydride Hydrolases↗

PubMed2006

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system.

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

Amino Acid Sequence↗

PubMed2005

Rotavirus glycoprotein NSP4 is a modulator of viral transcription in the infected cell.

The outer shell of the rotavirus triple-layered virion is lost during cell entry, yielding a double-layered particle (DLP) that directs synthesis of viral plus-strand RNAs. The plus-strand RNAs act as templates for synthesis of the segmented double-stranded RNA (dsRNA) genome in viral inclusion bodies (viroplasms). The viral endoplasmic reticulum (ER)-resident glycoprotein NSP4 recruits progeny DLPs formed in viroplasms to the ER, where the particles are converted to triple-layered particles (TLPs) via budding. In this study, we have used short interfering RNAs to probe the role of NSP4 in the viral life cycle. Our analysis showed that knockdown of NSP4 expression had no marked effect on the expression of other viral proteins or on the replication of the dsRNA genome segments. However, NSP4 loss of function suppressed viroplasm maturation and caused a maldistribution of nonstructural and structural proteins that normally accumulate in viroplasms. NSP4 loss of function also inhibited formation of packaged virus particles, instead inducing the accumulation of empty particles. Most significant was the observation that NSP4 knockdown led to dramatically increased levels of viral transcription late in the infection cycle. These findings point to a multifaceted role for NSP4 in virus replication, including influencing the development of viroplasms, linking genome packaging with particle assembly, and acting as a modulator of viral transcription. By recruiting transcriptionally active or potentially active DLPs to the ER for conversion to quiescent TLPs, NSP4 acts as a feedback inhibitor down-regulating viral transcription when adequate levels of plus-strand RNAs are available to allow for productive infection.

Animals↗

PubMed2004

Replication and transcription of the rotavirus genome.

Rotaviruses are an important cause of human morbidity and mortality, representing the primary pathogens responsible for acute dehydrating diarrhea in children under the age of 3. The infectious rotavirus particle is made up of three concentric layers of protein, and contains a genome consisting of eleven segments of double-stranded (ds)RNA. Upon infection, RNA polymerases associated with double-layered virus particles are activated, resulting in genome transcription and extrusion of the eleven viral mRNAs from such particles. The mRNAs not only direct protein synthesis, but also serve as templates for minus-strand synthesis to yield dsRNAs. Synthesis of the dsRNAs is an event that occurs following the gene-specific packaging of viral mRNAs into core-like assembly intermediates. Electron-dense cytoplasmic inclusions, termed viroplasms, function as sites of genome packaging and replication in the infected cell. Our understanding of key events in the viral life cycle has been advanced considerably by the development of cell-free systems that support mRNA synthesis from virion-derived double-layered particles and dsRNA synthesis from virion-derived core particles. The recent expression and purification of rotavirus recombinant proteins have also allowed progress to be made in defining the roles of viral proteins in genome replication and viroplasm formation. However, our efforts towards a full description of the viral life cycle, most notably an understanding of the events occurring during gene-specific packaging, remain hampered by the lack of a cell-free packaging system and a reverse genetics systems. The lack of a reverse genetics systems also confounds efforts towards the generation of molecular engineered second-generation vaccines.

Cell-Free System↗

PubMed2003

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2.

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (HØHØHØØ where Ø is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.

Adaptor Proteins, Signal Transducing↗