The morphology and composition of influenza A virus particles are not affected by low levels of M1 and M2 proteins in infected cells

Abstract

We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.

FIG. 1.

Representation of the influenza A virus vRNA promoter of the M segment in the corkscrew conformation. Conserved nucleotides in influenza A virus RNAs are shown in boldface. The introduced C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends, respectively, is shown. Base pairs are joined by lines. Conserved base pairs in the double-stranded region involving both the 3′ and 5′ ends of the RNA segment are boxed. The 5′-end numbers are distinguished by primes. Numbering of residues starts from the 3′ end and from the 5′ end. Start and stop codons are also shown. The new start and stop codons created by the 3′ mutation are indicated by an asterisk.

FIG. 2.

Growth curves of Mmut and WSN viruses and synthesis of viral proteins in infected cells. MDCK cells were infected at an MOI of 0.001 (A) and MOI of 1 (B) with WSN (♦) and Mmut (□) viruses. At the indicated time points, infectious particles present in the medium were titrated by plaque assay. (C) Patterns of protein expression in MDCK cells infected with Mmut and WSN viruses (MOI of 1). Cells were ³⁵S labeled for the indicated periods of time. Labeled mock-infected cells are also shown. Labeled proteins were separated on a 16.5% Tris-Tricine gel. Positions of the major viral proteins NP, M1, and NS1 are indicated on the right. (D) Detection of M1 and M2 proteins in infected cells by Western blotting with the antibody E10. (E) NP localization in virus-infected cells as determined by NP immunofluorescence. hpi, hours postinfection.

FIG. 3.

Protein composition, vRNA profile, and morphology of the Mmut virus particles. (A) Protein gel analysis of Mmut and WSN viruses. Purified viruses were either untreated (−) or treated with PNGase F (+), subjected to SDS-10% PAGE, and stained with Coomassie brilliant blue. Positions of proteins are indicated. BSA, bovine serum albumin. (B) Western blots of Mmut and WSN virions to detect HA, NP, M1, and M2 proteins. Volumes of virus preparations loaded on the gel are shown. (C) vRNA profile of WSN and Mmut viruses. The RNAs were extracted from purified virions, separated on a 2.8% polyacrylamide gel containing 7 M urea, and visualized by silver staining. The positions of RNAs that encode the polymerase proteins (Ps), HA, NP, NA, M1 and M2 proteins (M), and NS1 and NEP proteins (NS) are indicated. The position of 18S rRNA is also indicated. (D) Electron microscopy of purified Mmut and WSN viruses. Pictures were taken at ×47,000 magnification. Bars, 270 nm.