Research Article: Bipartite interface of the measles virus phosphoprotein X domain with the large polymerase protein regulates viral polymerase dynamics

Date Published: August 5, 2019

Publisher: Public Library of Science

Author(s): Venice Du Pont, Yi Jiang, Richard K. Plemper, Sean P.J. Whelan.

http://doi.org/10.1371/journal.ppat.1007995

Abstract

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility.

Partial Text

Non-segmented negative polarity RNA viruses comprise the etiological agents of some of the most devastating acute viral infections, such as Ebola virus, rabies virus, and, of the paramyxovirus family, Nipah virus and MeV. Common to all viruses in this order, the single-stranded RNA genomes are wrapped by nucleocapsid (N) proteins into helical ribonucleoprotein (RNP) complexes, and encapsidated RNAs are exclusively recognized as templates for transcription and replication by the viral RNA-dependent RNA-polymerases (RdRPs) complexes [1].

To dissect the contribution of individual MeV PCT domains to L binding, we first focused on OD. In MeV P expression plasmids, we either deleted this domain by removing residues 303–360 (OD structure up to residue 360 was resolved in [42]) or replaced this section up to residue 360 or 377 (end of defined heptad repeat motif), respectively, with the tetrameric mutant of the yeast general control protein 4 (GCN4) coiled-coil [43] (Fig 1B). The effect of these modifications on physical interaction of P with L was assessed through co-immunoprecipitation (co-IP) of the proteins from lysates of transiently transfected cells. All co-IPs were carried out with a C-terminally truncated form of L, L1708 lacking the L methyltransferase domain, that we have previously shown to be fully folding competent and capable of forming bioactive polymerase complexes when post-translationally combined with the corresponding C-terminal L fragment [44]. Co-IP efficiencies of L1708 or full-length L with P are indistinguishable (S1 Fig), but L1708 facilitates biochemical analysis of P-to-L binding due to a lower tendency to self-aggregate spontaneously [45].

This study demonstrates that distinct microdomains in MeV P are involved in efficient P hetero-oligomerization with L. We favor the view that these microdomains engage L through direct protein-protein contacts, although more indirect associations involving, for instance, host-derived adapter proteins cannot be excluded. Here, the term P:L interface will be used to refer to both direct and multi-component interactions between the P and L proteins. Based on a combination of biochemical and functional assays, we have pioneered a trans-complementation system to probe the stoichiometric requirements for productive P:L interactions. While trans-complementations were carried out in minireplicon assays, a recombinant MeV carrying a P trans-complementation pair in two tandem open reading frames could be recovered readily, efficiently proliferated with standard MeV-like growth profile, and was genetically stable over multiple passages. The absence of compensatory or reversion mutations, in particular, serves as a compelling indicator that the mutant recMeV does not experience strong selective pressure. These results confirm that trans-complementation restored all RdRP activities required for successful paramyxovirus replication. Thus assured of the physiological relevance of the strategy, the assay has advanced our mechanistic understanding of how paramyxovirus RdRp negotiates the encapsidated template in four areas:

 

Source:

http://doi.org/10.1371/journal.ppat.1007995

 

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