Molecular Determinants of Arenavirus Z Protein Homo-Oligomerization and L Polymerase Binding

Abstract

The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.

Multifunctional Nature of the Arenavirus RING Finger Protein Z

“…N-terminal myristoylation is also a conserved feature of all arenavirus Z proteins that is required for their biological functions [70,71]. The myristyl moiety facilitates Z membrane anchoring and intracellular targeting, Z self-assembly, and interaction of Z with other arenavirus proteins [71,72,73]. Consequently, treatment with myristic acid analogs leads to dose-dependent inhibition of Z-mediated release of VLPs and significantly reduces infectious virus particle production of several OW and NW arenaviruses [70,71,74].…”

“…Residues within the Z protein that are presumably involved in L binding have been identified by assessing the ability of various Z mutants to inhibit viral RNA synthesis. The inhibitory activity of LCMV and LASV Z requires an intact RING domain, whereas the N- and C-terminal residues are not essential for this function [73,89]. In particular, the Zn ++ -coordinating residues and a highly conserved tryptophan residue within the RING domain are essential for the strong inhibitory activity of Z on RNA synthesis [90].…”

Advanced Vaccine Candidates for Lassa Fever

“…There is no licensed vaccine for the prevention of LF and vaccine development efforts are hampered by both the high cost of non-human primate (NHP) animal models and biocontainment requirements (BSL-4) for study and development. LASV has the highest human impact of any of the hemorrhagic fever viruses (with the exception of Dengue fever) with an estimated 100,000-300,000 infections and 5,000-10,000 deaths annually in western Africa [1,2,3,4,5]. Based on prospective studies performed in four of the most affected countries, Guinea, Sierra-Leone, Liberia, and Nigeria, Richmond and Baglole [5] estimated that 59 million people are at risk of primary LASV infections with an annual incidence of disease as high as 3 million and as many as 67,000 deaths per year.…”

The involvement of epithelial cells in arenavirus-induced pathogenesis.

“…LASV is the most pathogenic of the OW arenaviruses, and is responsible for several hundred thousand infections, and thousands of deaths annually in Western Africa [45,[78][79][80].…”

“…Annually in Western Africa, there are several hundred thousand clinical LASV infections, and thousands of deaths due to LF [45,[78][79][80]. Although LASV causes a significant number of deaths, the majority of infections are apparently subclinical, or not severe enough to warrant emergency medical intervention, as over 45% of the population in endemic regions is seropositive for LASV; however, why some of the population develop disease and others do not it is not well understood [81].…”