Myristoylation of the RING Finger Z Protein Is Essential for Arenavirus Budding

Abstract

The arenavirus small RING finger Z protein is the main driving force of arenavirus budding. The primary structure of Z is devoid of hydrophobic transmembrane domains, but both lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus Z proteins accumulate near the inner surface of the plasma membrane and are strongly membrane associated. All known arenavirus Z proteins contain a glycine (G) at position 2, which is a potential acceptor site for a myristoyl moiety. Metabolic labeling showed incorporation of [ 3 H]myristic acid by wild-type Z protein but not by the G2A mutant. The mutation G2A eliminated Z-mediated budding. Likewise, treatment with the myristoylation inhibitor 2-hydroxymyristic acid inhibited Z-mediated budding, eliminated formation of virus-like particles, and caused a dramatic reduction in virus production in LCMVinfected cells. Budding activity was restored in G2A mutant Z proteins by the addition of the myristoylation domain of the tyrosine protein kinase Src to their N termini. These findings indicate N-terminal myristoylation of Z plays a key role in arenavirus budding.

Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest

“…Rib inhibits arenavirus replication via mechanisms that have not been entirely elucidated but most likely involve the targeting of different steps of virus RNA synthesis (21). However, 2-OHM inhibits myristoylation of Z, which is required for arenavirus budding (22). Based on their mechanisms of action, we predicted that Rib would similarly inhibit production of virus progeny upon infection at low or high moi, whereas the inhibitory effect of 2-OHM would be expected to be more pronounced in infections initiated at low moi involving multiple rounds of cell infection required for virus propagation within the cell population.…”

A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles

“…We recently demonstrated that the PPXY late domain in LCMV Z is not absolutely required for the production of infectious LCMV virions (Ziegler et al, 2016). Provided that the only other motif in Z with a known role in budding activity is the myristoylation site at the glycine at position 2 (Figs 1c and 2d) (Perez et al, 2004;Strecker et al, 2006), our finding here expands the functional repertoire of motifs in LCMV Z that regulate the efficiency of infectious virus release. Further, we have built upon our previous findings regarding the PPXY late domain (Ziegler et al, 2016) by showing that S41 also serves as a key regulator of DI particle formation.…”

“…To determine whether the reduction in infectious titre of the phosphomimetic S41D virus was due to decreased virus budding and release, we employed a Z VLP release assay as previously described (Ziegler et al, 2016). As a control, we also included the LCMV Z G2A mutant, which exhibits a pronounced defect in VLP formation due to its inability to be myristoylated at this glycine residue (Perez et al, 2004;Strecker et al, 2006). This experiment demonstrated that the budding efficiency of the phosphomimetic Z-S41D was reduced~60 %, while the budding activity of the nonphosphorylatable S41A was not different from WT (Fig.…”

“…(d) The budding activity of WT or S41-mutant LCMV Z proteins was measured by a VLP release assay. The LCMV Z G2A mutant, which has a budding defect due to its inability to be myristoylated, was included as a control (Perez et al, 2004). The results shown represent the mean±SEM from three independent experiments.…”

Lymphocytic choriomeningitis virus: ways to establish and maintain non-cytolytic persistent infection

“…It is the main driving force of virion budding (Salvato et al, 1992;Strecker et al, 2003). It contains a conserved RING-fi nger domain fl anked by an Nterminal hydrophobic domain with myristoylation and phosphorylation site (Perez et al, 2004). Th e C-terminal portion of ZP contains proline-rich motifs that have been identifi ed as late motifs in matrix proteins (Eichler et al, 2004;Freed, 2002).…”