Z alone could direct VLP`s formation in Sf9 insect cells through budding

BORIO, C.S.; STEPHAN, B.I.; ISERTE, J.A.; GOÑI, S.E.; GHIRINGHELLI, P.D.; LOZANO, M.E.

Villa Carlos Paz, Córdoba

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Junín virus, the etiological agent of Argentine hemorrhagic fever, belongs to the Arenaviridae family. This family has 23 recognized species with an ambisense-bipartite ssRNA genome. The enveloped virions include non-equimolar amounts of each genomic RNA, named L (ca. 7200 nt; L and Z proteins), and S (ca. 3500 nt; N and GPC proteins). The Z protein is a structural component of the virion that interacts with several proteins in infected cells, interfering with the normal protein expression pattern. We analyzed Z amino acid sequences and divided it into three domains. The C-terminal region contains a domain involved in budding process. The core region comprises 8 amino acids corresponding to the ring finger structure. The N-terminal region, contains a conserved G at position 2, that would be myristoilated. This mediates protein localization in the plasma membrane, essential for viral particle formation. We expressed and purified Z protein in bacterial, insect and mammalian cells, from Candid#1 strain, and obtained a polyclonal serum anti-Z fused to thiorredoxin, used for immuno detection techniques. Insect cells-expressed His-tag-Z protein shows the lost of the tag after it`s expression a result agreeing with myristoilation. We verified the VLP´s formation, in insect and mammalian cells as consequence of the Z expression alone, by differential ultracentrifugation and western blotting.