ROLES OF NSP4, VP4 AND VP7 PROTEINS IN THE ROTAVIRUS MORPHOGENESIS. José Luis Zambrano R . (1), Mirtha Romano (2), Esmeralda Vizzi (1), Marie Christine Ruiz (3), Fabian Michelangeli (3), Ferdinando Liprandi (1), Juan Ernesto Ludert (1). (1) Laboratorio de Biología de Virus, Centro de Microbiología y Biología Celular. Instituto Venezolano de Investigaciones Científicas; (2) Servicio de Microscopía Electrónica. Centro de Microbiología y Biología Celular. Instituto Venezolano de Investigaciones Científicas; (3) Laboratorio de Fisiología Gastrointestinal. Centro de Biofísica y Bioquímica. Instituto Venezolano de Investigaciones Científicas. Caracas, Venezuela. Apartado Postal 1020-A. E-mail: jzambran@ivic.ve Rotaviruses are the leading cause of several gastroenteritis in infant and young children worldwide. The rotavirus, a double-stranded RNA and non-enveloped viruses have three concentric layers of protein with icosahedrical architecture. The new particles of the virus are synthesized in the cytoplasm in a large cytoplasmatic inclusion bodies, termed viroplasm [1, 2] rich in nucleic acid and proteins that serves as viral factories. The final maturation of the virion including: i. a budding process of the new particles (DLP) to the endoplasmatic reticulum (ER), facilitated by the non structural protein NSP4 [3, 4]; ii. a enveloped transiently intermediated state [5] iii. elimination of the temporal enveloped [5], and iv. acquisition of the outer protein layer (TLP) formed by the structural proteins VP4 and VP7 [6]. The aim of this study was determinate the rol of the NSP4, VP4 and VP7 protein in the rotavirus morphogenesis. MA104 were transfected with specific siRNA for silencing of the expression of NSP4, VP4 and VP7 rotavirus proteins. At 36 h.p.t the cells were infected with rotavirus DxRRV (m.o.i 10). At 7 h.p.i., the cells were harvested and fixed with 0.2 % glutaraldehyde in 0.1 M cocadilate buffer (pH 6.7) and post-fixed with 2 % OsO 4 . Fixed cells were embedded into Epon resine. Ultrathin sections were dyed with 2% uranil acetate and examined with a Phillips CM10 transmission electron microscope. In control cells (Figure A), show a normal rotavirus replicative cycle: viroplasm attached to the ER and the DLP budding process was observed (1A), enveloped transiently into the ER (1B) and the further elimination with the VP4 and VP7 proteins outer layer assembly to conform the mature and infective TLP particle (1C). Silencing of the expression of the non structural protein NSP4, avoid the viroplasm-ER attachment (Figure B) and immature DLP were observed free in the cytoplasm (1B). When VP7 protein was not expressed (Figure C), the transiently enveloped was retained (1C) and the rotavirus replicative cycle was abolished. A very different situation was observed when the structural protein VP4 was silencing (Figure D). All the first stages showed in control cells were observed: viroplasm-ER attachment (1D), budding process (2D) and transiently enveloped elimination, but a newly paracrystalline aggregation of the viral particles was observed (3D). These aggregations of the viral particles prevent the virion successful maturation. Our results suggest the key roles of the NSP4, VP7 and VP4 proteins at different stage of the viral morphogenesis: NSP4 protein was necessary for the viroplasm-ER attachment and for the initiation of the budding process; VP7 protein was essential for the elimination of enveloped transiently, and VP4 protein was required for the correct folding of the rotavirus outer layer. References: [1] BC Altenburg, et al. J Gen Virol. (1980). 46, 75. [2] RA González, et al. Arch Virol. (2000). 145, 1963. [3] KS Au, et al. J. Virol. (1993). 63, 4553. [4] P Tian, et al. Arch Virol Suppl. (1996). 12, 69. [5] JB Pasavento, et al. Curr Top Microbiol Inmunol. (2006). 309, 189. 259 Cusco-Peru September 2007 Acta Microscopica Vol 16 No1-2,(Supp.2)2007