Our study provides unequivocal evidence for the essential roles of the invariant residues of arenaviral Z protein in infectious viral replication. These residues, including G2, the zinc-binding C and H residues within the RING domain, and L78 and P79 (numbered according to PICV Z protein), are absolutely conserved among all known Z proteins. Previous studies have demonstrated some of these residues' critical roles in VLP formation and viral RNA regulation (3
). Our study, however, establishes their essential roles in the context of infectious virus life cycle, since alanine substitutions at these residues have resulted in nonproductive PICV virus production (). The proline-rich L domain can tolerate mutations to some levels; however, these mutations have dramatically altered viral plaque formation and significantly reduced viral growth ability, suggesting an important role of the intact L domain in viral replication. Our comprehensive analyses of key residues of arenaviral Z proteins have also provided some novel insights into their mechanistic roles in virus budding, nucleocapsid incorporation, and viral RNA regulation, as discussed below.
An essential biological role of the Z protein in arenavirus infection is the Z-mediated virus budding. From both previous publications (19
) and the present study (), the G2 myristylation site is indispensable for the Z protein-mediated budding activity, which most likely explains the vital role of G2 in arenavirus infection (). Also important in mediating Z budding is its C-terminal L domain(s) (18
). The L domain consists of a stretch of conserved amino acid residues that interact with components of the ESCRT pathway to mediate membrane budding. Thus far, three classes of viral L domains have been defined, PT/SAP, LxxLF or YPXL, and PPxY, which interact with Tsg101, ALIX, and NEdd4-like HECT ubiquitin ligases, respectively (10
). These L domains are functionally interchangeable (26
). Some arenaviral Z proteins, such as LASV Z, contain two separate L domains PTAPP and PPPYSP, whereas others contain a proline-rich region that seems to consist of two overlapping L domains (e.g., PSAPPYEP for PICV) or just one L domain (e.g., PTAPPP for Junin) (). Although dicodon mutation of the Proline residues in PSAPPYEP of PICV Z protein has minor effect on its self-budding activity (, lanes 8 to 10), possibly due to the overexpression system, they have significantly altered viral plaque morphology and reduced viral growth (), suggesting that the intact L domain(s) of arenaviral Z proteins are required for efficient virus budding and replication.
The mechanism governing the incorporation of nucleocapsids into arenavirus virions is largely unknown. It has been proposed that the interaction between Z and NP is required to recruit and incorporate the nucleocapsids into budding virions at the cell membrane (4
). A recent study suggests that ALIX, a component of ESCRT network that interacts with L domain LxxLF or YPXL, is required for NP incorporation into Mopeia virus Z-induced VLPs by interacting with both Z and NP (22
). Our study did not identify a single residue/domain of Z that is indispensable for NP incorporation (), but suggests that the LPTK site, and possibly, the RING domain, may play a more important role.
The central RING domain is a zinc-binding motif with invariant C and H residues that bind two zinc cations. Although we have shown that the zinc-binding residues are absolutely essential for arenavirus replication (), the functional role(s) of the RING domain in arenavirus biology is less clear. It seems unlikely that the lethal phenotype observed for all RING domain mutations is due to the abolishment of either Z budding or NP incorporation, because these Z mutations show differential effects on Z budding activity () and NP incorporation efficiency (). On the other hand, all of the RING domain mutations completely abolish the Z protein's ability to inhibit viral RNA synthesis (), although it remains unclear how Z-mediated inhibition of viral RNA synthesis is vital for arenavirus infection. A possible hypothesis is that this inhibition is necessary in order to initiate the viral assembly step. Further studies are required to fully understand the biological significance of Z-mediated viral RNA inhibition in arenavirus life cycle.
We have demonstrated an essential role for L78 or P79 (numbered according to PICV Z protein) in infectious viral production. Using the PICV reverse genetics system, we have shown that both L→A and P→A mutations in the Z protein lead to a complete loss of viral infectivity (). This is consistent with a recent study by Capul et al. (3
) showing that both L→A and P→A mutations of LASV Z protein strongly reduce VLP infectivity. Their functional mechanisms in infectious viral replication, however, are less clear. We and other researchers have shown that mutagenesis of the invariant L78 or P79 residue does not affect Z budding activity (3
; the present study). Furthermore, both lethal (L78A and P80A) and nonlethal mutants (T81A and K82A) in the LPTK motif led to an ~50% decrease in NP incorporation. Therefore, we do not believe that the indispensable role of L and P residues in the viral life cycle is to mediate the NP incorporation into viral particles. On the other hand, we () and the Capul study (3
) have shown that both L→A and P→A mutations abolish Z-mediated inhibition of viral RNA synthesis, which may explain their inability to support infectious viral replication as discussed above for the RING domain mutants. Nevertheless, the exact functional mechanisms of the invariant L and P residues in arenaviral infectious life cycle remain to be fully elucidated.
Finally, our study has characterized the molecular mechanisms of both LASV and PICV Z protein in budding () and RNA regulation (). Most of the conserved residues/domains have similar functional mechanisms in the different Z proteins, such as the role of G2 in budding, and that of RING domain and LPTK in RNA inhibition. There are, however, some interesting variations. A notable one is the role of G2 in blocking viral RNA synthesis (). The G2 residue is indispensable for PICV Z-mediated viral RNA inhibition but is unnecessary for LASV Z. A previous study has shown that the Z protein of LCMV, another Old World arenavirus, does not require the N-terminal residues 1 to 16 (including G2) to inhibit viral RNA synthesis (5
). It remains to be determined whether the G2 residue of other arenavirus proteins plays an important role in viral RNA regulation. In addition, a recent study using Machupo virus polymerase complex in an in vitro
polymerase assay has suggested that Z directly interacts with the L polymerase protein to block the early steps of viral RNA synthesis in a species-specific manner (12
). Therefore, the functional mechanism of Z in viral RNA regulation requires further characterization in specific arenavirus species.
In summary, our studies have provided evidence for the essential roles of the conserved residues (domains) of the Z protein in the infectious arenavirus life cycle. In addition, our studies have also provided some novel insights into the functional mechanisms of the conserved residues (domains), including the poorly characterized LP(TK) residues, of the Z protein in viral infectivity. These studies may lead to the development of novel antivirals targeting the essential Z protein in order to treat arenavirus-induced diseases.