Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes

Abstract

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.The influenza A virus M1 protein is the most abundant structural component of the virion, and electron microscopy studies of influenza virus A particles show that M1 forms a shell at the internal surface of the viral membrane (6, 39, 47). The functions of M1 have been studied extensively, and it has been implicated in a variety of roles in the virus life cycle that include RNA and RNP binding (4,5,14,41,43,51,52,55,57), transcription inhibition (5, 14, 52, 58, 62), and control of RNP nuclear import and export (8,21,34,35,53,54). In addition, cell fractionation experiments with expressed M1 protein or virus-infected cells show it to exist in both soluble and membrane-bound forms. There are examples in which coexpression of the viral glycoproteins was found to stimulate membrane association overall (15), but this was not noted in other reports (30,59). It is also thought that M1 recruitment to lipid raft microdomains on the apical surface of polarized epithelial cells, the site of influenza virus budding, is stimulated by the presence of the intact glycoproteins (1, 3, 61). Taken together, most of the results support the long held hypothesis that M1 can associate with RNPs on one hand and viral glycoproteins on the other and plays a prominent role in virus assembly.Analogous matrix proteins are considered important for the process of assembly and budd...

Structural organization of a filamentous influenza A virus

“…Virions showing even more diversity of shape and width still show evidence of a similar, locally flat 2D lattice of the M1 protein, consistent with M1 being able to polymerize with a wide range of curvatures. A previously described mutant of the M1 protein (K102A) of A/WSN/33, obtained by reverse genetics, transforms a non-filamentous strain to a filamentous phenotype (28). In Fig.…”

“…We have shown that a highly ordered, helical organization of the matrix layer is associated with the cylindrical morphology of filamentous and capsule-shaped Udorn influenza virus. Previously, the M1 gene segment has been identified as a genetic determinant of filamentous morphology (20,28,32,33). Although the M1 protein alone was reported to be essential for virus-like particle (VLP) formation and capable of forming intracellular tubes (34), it now appears that viral membrane proteins are required for budding (35).…”

“…The sequence of the Udorn M1 protein likely confers the filamentous phenotype by stabilizing specific helical contacts in the matrix layer (28). Length variation in Udorn particles may reflect a competition between extension of the M1 helix and the completion of budding ("pinching off") and release.…”

“…A/Udorn/72 (a gift from R. Compans, Emory University School of Medicine, Atlanta, GA), A/WSN/33, and M1 mutant K102A (a gift from D. Steinhauer, Emory University School of Medicine, Atlanta, GA) were grown in Madin-Darby canine kidney (MDCK) cells and purified over sucrose gradients as previously described (28). A/Aichi/68 X-31 virus was grown in embryonated hens' eggs and purified over sucrose gradients as previously described (47).…”

Identification of Conserved Peptides Comprising Multiple T Cell Epitopes of Matrix 1 Protein in H1N1 Influenza Virus

“…Influenza virus exists in two different morphological forms: spherical (*100 nm diameter) and filamentous (*100 nm · 2-20 lm) virions (18). The filamentous trait of influenza virus has been traced to helix 6 domains of the M1 protein (8). The helix 6 domain (91-105 amino acids) of the M1 protein has been recognized as the multifunctional domain acting as a nuclear localization signal (NLS) for the translocation of the M1 protein in the nucleus and transcription inhibition motifs.…”