Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association

Abstract

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.A ll members of the Orthomyxoviridae family, including the influenza viruses A-D, thogotovirus, and isavirus, encode a matrix protein called M1. In the influenza A virus, M1 is produced by a colinear transcript made from the gene segment 7 (1). As one of the most abundantly made viral proteins, the influenza A virus M1 plays multiple roles during the virus life cycle. Upon viral entry, the acidification of the viral interior in the endosome weakens the interaction between M1 and the viral ribonucleoprotein complexes (vRNPs), thus allowing M1-free vRNPs to be imported to the nucleus for viral RNA replication and transcription (2, 3). As infection proceeds, newly synthesized M1 enters the nucleus to mediate the nuclear export of nascent vRNPs. A "daisy-chain" complex is formed with the vRNP binding to the C-terminal domain of M1 (4) and the N-terminal domain of M1 interacting with the nuclear export protein (NEP), also called NS2. Through its nuclear export signal (NES), NEP is specifically recognized by the cellular exportin Crm1, which then facilitates the transport of vRNPs across the nuclear membrane to the cytoplasm in a RanGTP-dependent manner (5). It has been shown that M1 binding to vRNP in the nucleus is able to block mRNA transcription (4, 6). In the cytosol, M1 interacts with the cytoplasmic tails of the glycoproteins HA and NA. Such interactions promote M1 association with lipid raft membranes and...

Structural Determinants of the Interactions Between Influenza A Virus Matrix Protein M1 and Lipid Membranes

“…Furthermore, protein–protein and protein– lipid interactions occurring during membrane binding of M1 are accompanied by a moderate but significant alteration in secondary structure. These findings support a model of M1–M1 interactions that we and others have discussed [21, 32, 38], according to which binding of M1 to the PM (mediated by the N-terminal domain) could affect the structure or the chain dynamics of the protein (specifically of the C-terminal domain), thus exposing or stabilizing the interfaces for M1 self-association [32] and, possibly, vRNP binding [21].…”

“…While crystal structures of the relatively compact M1 N-terminal domain have been solved at pH 4.0 and 7.0, both the structure and the exact orientation of the more flexible and disordered C-terminal domain within the full-length protein remain unclear [33-37]. A recently published crystal structure of the matrix protein from another orthomyxovirus, the infectious salmon anaemia virus (ISAV), provides first clues on the 3D structure of full-length IAV M1 [38].…”

The native structure of the full-length, assembled influenza A virus matrix protein, M1

“…A trans-interface between NTD and CTD is present within the crystal packing of the ISAV matrix protein (Extended data Fig. 3d) 13 .…”

“…There are no structures available for the CTD of M1. A structure of the full-length matrix protein is available from an orthomyxovirus Infectious Salmon Anemia Virus (ISAV), however substantial divergence (18% sequence identity) between ISAV and IAV matrix proteins makes it unclear how relevant the ISAV matrix protein structure is to IAV M1 13 . Classical electron microscopy suggests that M1 forms a helical arrangement of fibers in both filamentous 14,15 and spherical virions 16 , but how M1 oligomerizes and is arranged to form the viral endoskeleton is unknown.…”

Continuing evolution of H6N2 influenza A virus in South African chickens and the implications for diagnosis and control

“…Although this phenomenon was noted previously [23], the reason for this is still not understood. The M1 protein interacts with the HA and NA glycoproteins during assembly of the virus particle [37], therefore the co-assortment of these three segments might reflect specific structural complementation in the proteins they encode, but further investigations are required for clarity. The surface glycoproteins HA and NA are constantly exposed to the host immune system and Rauff et al [23] previously reported that sub-lineage I mutates at the higher rate of 7.7 x 10 -3 nucleotide substitutions per site per year compared to 4.05 x 10 -3 for sub-lineage II, attributed to vaccine pressure.…”