The RING domain and the L79 residue of Z protein are involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious chimeric arenavirus-like particles.
Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.
Casabona
Juan Cruz
JC
Centro de Virología Animal (CEVAN), Instituto de Ciencia y Tecnología Dr Cesar Milstein, Consejo Nacional de Ciencia y Tecnología (CONICET), Saladillo 2468, C1440FFX Ciudad Autónoma de Buenos Aires, Argentina.
Levingston Macleod
Jesica M
JM
Loureiro
Maria Eugenia
ME
Gomez
Guillermo A
GA
Lopez
Nora
N
D000595:Amino Acid Sequence; D000818:Animals; D001117:Arenaviridae Infections / Q000378:metabolism / Q000821:virology*; D006477:Arenaviruses, New World / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054829:RING Finger Domains; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D006023:Glycoproteins; D014764:Viral Proteins