Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) surrounded by three protein layers. The virus contains an RNA-dependent RNA polymerase that synthesizes RNA transcripts corresponding to all segments of the viral genome. These transcripts direct the synthesis of the viral proteins and also serve as templates for the synthesis of the complementary strand to form the dsRNA genome. In this work, we analyzed the kinetics of transcription and replication of the viral genome throughout the replication cycle of the virus using quantitative reverse transcription-PCR. The role of the proteins that form doublelayered particles ([DLPs] VP1, VP2, VP3, and VP6) in replication and transcription of the viral genome was analyzed by silencing their expression in rotavirus-infected cells. All of them were shown to be essential for the replication of the dsRNA genome since in their absence there was little synthesis of viral mRNA and dsRNA. The characterization of the kinetics of RNA transcription and replication of the viral genome under conditions where these proteins were silenced provided direct evidence for a second round of transcription during the replication of the virus. Interestingly, despite the decrease in mRNA accumulation when any of the four proteins was silenced, the synthesis of viral proteins decreased when VP2 and VP6 were knocked down, whereas the absence of VP1 and VP3 did not have a severe impact on viral protein synthesis. Characterization of viral particle assembly in the absence of VP1 and VP3 showed that while the formation of triple-layered particles and DLPs was decreased, the amount of assembled lower-density particles, often referred to as empty particles, was not different from the amount in control-infected cells, suggesting that viral particles can assemble in the absence of either VP1 or VP3.The family Reoviridae includes viruses that have a genome composed of 9 to 12 segments of double-stranded RNA (dsRNA). Rotaviruses belong to the most medically significant genus of the family since they are the main cause of infantile gastroenteritis, causing approximately 500,000 deaths per year in children less than 5 years of age (23). These viruses have a genome composed of 11 segments of dsRNA, which is enclosed in a capsid formed by three concentric layers of protein (33). The innermost layer, formed by VP2, contains the viral genome and 12 copies each of the virus RNA-dependent RNA polymerase (RdRP; VP1) and the guanylyltransferase and methylase enzyme (VP3); these viral elements constitute the core of the virus. The addition of VP6 on top of the VP2 layer produces double-layered particles (DLPs). The outermost layer, characteristic of infectious, triple-layered particles (TLPs), is composed of two proteins, VP4 and VP7.During or shortly after cell entry, the infecting TLP uncoats, loosing the two proteins of the outer layer and yielding a DLP, which is transcriptionally active (14). The nascent transcripts are extruded into the cell's cytoplasm through channels located ne...

The in vitro characteristics of human rotavirus transcription have been examined. The virus has an associated RNA polymerase activity which was activated after a heat shock treatment. The enzyme required the presence of the four ribonucleoside triphosphates and a divalent cation (Mg2"), and it required an optimum pH of 8.5. The polymerase was activated by monovalent salts and inhibited by Na PPi. The addition of actinomycin D, a-amanitin, or rifampin did not inhibit the polymerase activity. After thermal shock of the virus, at least eight different RNA species were synthesized which may correspond to independent transcripts. Transcription also requires a hydrolyzable form of ATP. Analogs such as ,B,y-imido ATP or f,By-methylene ATP were inhibitory, whereas others, such as the f,ty-imido or 'methylene analogs of CTP, UTP, or GTP, were not inhibitory. This suggests that ATP is related to reactions other than polymerization, probably to initiation or elongation of RNA molecules, as has been described for vesicular stomatitis virus or vaccinia virus.