The Intracellular Distribution of Influenza Virus Matrix Protein and Nucleoprotein in Infected Cells and Their Relationship to Haemagglutinin in the Plasma Membrane

Abstract

SUMMARYPre-and post-embedding immune electron microscopy techniques employing ferritin and large and small gold markers to detect cell surface and intracellular antigens respectively, have been combined in a study of influenza virus-infected cells. This has permitted, for the first time, the simultaneous detection of intracellular virus matrix protein (M), nucleoprotein (NP) and membrane haemagglutinin (HA). The technique facilitated an investigation of the possible physical interrelationship between these three proteins both in the infected cell, and on the infected cell membrane. Electron-dense bodies uniformly labelled by antibody to M protein were observed in the nucleus and cytoplasm. Similarly, NP was detected in both the nucleus and cytoplasm. Approximately 509/oo of the nuclear N P was located in close proximity to the M protein-containing dense bodies but mainly on the perimeter of the structures. A similar relationship of NP to the M-containing dense bodies was observed in the cytoplasm. M protein and NP were readily detected in sections of budding virions. Labelling of these proteins was also observed on the cytoplasmic face of the plasma membrane but the density of labelling only occasionally approached that of newly formed virions. These findings suggest that budding occurs very quickly after the internal proteins arrive at the plasma membrane. Double labelling experiments on the cell surface indicate that NP and HA behave as independent molecules and do not form tight complexes with each other.

Antigenic Characterization of the Nucleoprotein and Matrix Protein of Influenza C Virus with Monoclonal Antibodies

“…It is generally accepted that the NP protein of influenza A virus accumulates in the nucleus (Watson & Coons, 1954;Lui, 1955). Although there is contradictory evidence as to whether the M 1 protein enters the nucleus, several recent reports clearly demonstrated its migration to the nucleus (Maeno et al, 1977;Beesley & Campbell, 1984;Patterson et al, 1988;Bucher et al, 1989), and one such report described nuclear staining of M1 protein which was characterized by more intense staining of the nucleoli, whereas nuclear staining of NP is characterized by the absence of stain from the nucleoli (Bucher et al, 1989). The association of M protein with the nucleoli was observed in HMV-II cells infected with the influenza C/Yamagata/1/88 strain.…”

“…The intracellular location of M1 is still controversial, with some reports describing its detection in both the nucleus and the cytoplasm (Gregoriades, 1973;Oxford & Schild, 1975;Hay & Skehel, 1975;Maeno et al, 1977;Patterson et al, 1988;Bucher et al, 1989) whereas others claim that it is absent from the nucleus (Lazarowitz et al, 1971;Krug & Etkind, 1973). At present, no information is available concerning the intracellular distribution of NP and the M protein during the replication cycle of influenza C virus.…”

Mutations in Influenza Virus M1 CCHH, the Putative Zinc Finger Motif, Cause Attenuation in Mice and Protect Mice against Lethal Influenza Virus Infection

“…In addition, it has recently been shown that the M1 protein contains a novel L domain involved in virus budding (21,22). These processes require the M1 protein to interact with the viral glycoproteins HA and NA (1), viral RNP (2,6,46,50,51,53,75), and other viral and host proteins. M1 associates with a number of cellular proteins, including histones (12,76), RACK-1 (cellular receptor of activated C kinase 1) (49), nucleosomes (12), VPS28 (vacuolar protein sorting 28), Cdc42 (cell division cycle 42) (21), protein kinase C (49), extracellular signalregulated kinase (48), and possibly other host proteins.…”

Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

“…Therefore, we postulate that the intracellular tubular aggregates detected in transfected cells are exclusively made up of M1 proteins. We are aware that amorphous M1-containing electron-dense bodies have been detected in influenza virus-infected cells (36), but it should be mentioned that these aggregates do not bear any resemblance to the tubular structures described here. Moreover, to our knowledge, filamentous structures have never been described in cells expressing a recombinant influenza virus M1 protein.…”