Role of the CM2 Protein in Influenza C Virus Replication: Analyses of Recombinant Viruses possessing CM2 Mutants

Yasushi Muraki

doi:10.4172/2153-0769.s1-003

Abstract

CM2 is the second membrane protein of influenza C virus. In the present study, to investigate the role(s) of CM2 in the virus replication, we generated recombinant influenza C viruses, rC65A and rN11A, which lack the palmitoylatioin and glycosylation sites of CM2, respectively. The rC65A virus grew as efficiently as the recombinant wild-type (rWT) virus did, whereas the rN11A grew less efficiently than the rWT virus. To study the difference more precisely, we generated influenza C virus-like particles (VLPs) lacking the CM2 glycosylation site (N11A-VLPs) and examined the VLPs and VLP-infected cells. As a result, the N11A-VLPs contain approximately 13% of the virus RNA found in wildtype VLPs (WT-VLPs), and N11A-VLP-infected HMV-II cells exhibited reduced reporter gene expression compared with that of WT-VLP-infected cells (WT : N11A = 1:0.1). Thus, supportive evidence was obtained that CM2 is involved in packaging and uncoating processes and that observed growth difference between rWT and rN11A can be attributed to the difference in the CM2 function.

Highlights

The RNA segment 6 (M gene) of the influenza C/Ann Arbor/1/50 (AA/50) strain is 1,180 nucleotides in length and encodes a 242-amino acid matrix protein (M1) and a 374-amino acid P42 from the spliced and unspliced mRNA, respectively [1,2]
The radioimmunoprecipitation experiment using the Monoclonal antibodies (MAbs) and anti-CM2 serum revealed that i) the synthesis and maturation of the HEF, NP and M1 proteins exhibited no significant differences between the recombinant wild-type (rWT)- and rN11A-infected cells and ii) no glycosylated forms of CM2 (CM2a and CM2b) were detected in the rN11A-infected cells (Figure 1)
The rWT or rN11A viruses were infected to HMV-II cells at an multiplicity of infection (MOI) of 5 and incubated at 33°C for up to 120 hrs

Summary

Introduction

The RNA segment 6 (M gene) of the influenza C/Ann Arbor/1/50 (AA/50) strain is 1,180 nucleotides in length and encodes a 242-amino acid matrix protein (M1) and a 374-amino acid P42 from the spliced and unspliced mRNA, respectively [1,2]. CM2 is a type III integral membrane protein that is oriented in membranes with a 23-amino-acid N-terminal extracellular domain, a 23-amino-acid transmembrane domain and a 69-amino-acid C-terminal cytoplasmic domain [7,8]. It is modified by N-glycosylation at an asparagine residue 11, and as a result, three forms of CM2 with different electrophoretic mobilities (CM2o, CM2a, and CM2b) are detected in infected cells [7,8]. A mannose-rich oligosaccharide core is added to unglycosylated CM2o to form CM2a, and the maturation of the carbohydrate chain from the high mannose type to the complex type converts CM2a into CM2b, the latter of which is modified by addition of polylactosaminoglycan

Methods

Results

Conclusion