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

Abstract

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.

Genetic Variation In Vitro and In Vivo of an Attenuated Lassa Vaccine Candidate

“…The selected mutations were more frequently seen in NP than in the L protein. This could be due to NP's variety of functions and interactions during the viral replication cycle: it encapsidates the genome segments, interacts with L protein to form the RNP core for RNA transcription, associates with Z protein for viral assembly, plays an important role in suppressing the innate immune response, and has exonuclease and nucleotide binding activities (31)(32)(33)(34)(35)(36)(37)(38). In contrast to NP, the L protein is less immunogenic, and its RdRp activity has to be more conservative.…”

Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production

“…proteins could modulate Z-mediated VLP production and affect the efficiency of VLP production (Casabona et al, 2009;Groseth et al, 2010;Shtanko et al, 2010;Wang et al, 2012). Therefore, we propose that co-expression of GPC together with Z is necessary to mimic LASV budding.…”

Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

“…Thus, RNA-free NP seems to exist exclusively as a trimer, which presumably has biological relevance. Besides being the building block of ribonucleoproteins, NP has additional functions during the life cycle of the virus, such as interaction with L and Z proteins and cellular proteins, budding of particles, suppression of the interferon response, exoribonuclease activity, and nucleotide binding (15)(16)(17)(18)(19)(20)(21)(22)(23). Some of these or other so far unknown functions of NP might be associated specifically with the trimeric configuration.…”

“…NP is the major component of the viral ribonucleoprotein complex. It physically associates with itself as well as with L and Z proteins (15)(16)(17)(18). The association with Z protein and ALIX/AIP1, an ESCRT-associated host protein, is important for incorporation of NP into virus-like particles (15,(17)(18)(19).…”