The critical role of M1, the most abundant protein in the virus particle, in influenza virus assembly and budding is undisputed. Results presented in this report indicate that both viral glycoproteins HA and NA affect the membrane association of M1 proteins, thereby providing evidence for the interaction of M1 with HA and NA. This conclusion was further strengthened by the requirement for the homologous transmembrane domain and cytoplasmic tail of HA in detergent resistance of the membrane-bound M1. Earlier attempts to demonstrate the interaction between M1 and influenza virus glycoproteins showing increased membrane association of M1 protein in the presence of homologous viral glycoproteins yielded conflicting results (5
), which can be attributed to significant variation (15 to 60%) in the intrinsic membrane-binding ability of M1 protein expressed alone. Experimental factors including the expression system used and relative ratios of M1 to glycoproteins present in coexpressing cells as well as the process of cell disruption used in releasing membranes and preparing the 4K supernatant may have contributed to these variations. To overcome these difficulties, we designed an assay which would eliminate the membrane association of M1 protein expressed alone without eliminating the membrane association of M1 from the M1-glycoprotein(s) interactions.
Influenza virus transmembrane proteins are sorted to the apical plasma membrane, the budding site of influenza viruses in polarized epithelial cells. Many of these apical proteins including HA and NA have been shown to preferentially cluster on the lipid rafts enriched in cholesterol and glycosphingolipids during their transport from the trans-Golgi membrane to the plasma membrane (2
), and this interaction of apical proteins with lipid rafts occurs in both polarized and nonpolarized cells (38
). Furthermore, we and others have shown that the transmembrane domains of influenza virus NA and HA provide an apical determinant and associate with TX-100-resistant lipid rafts (2
). However, M1, a cytoplasmic protein, which is not transported by the exocytic pathway, is not expected to be raft associated and TX-100 detergent resistant unless it binds to another raft-associated protein, as has been shown for a number of signaling molecules (36
). Therefore, TX-100 detergent treatment essentially eliminates all lipid-protein interactions except for those proteins present in cholesterol- and glycosphingolipid-enriched membranes, and these detergent-resistant membrane-bound proteins will float to the top of the gradient. However, such detergent extraction of membranes should not be confused with TX-100 treatment used for assaying cytoskeleton-protein interactions (25
) since cytoskeleton-protein interactions are resistant to a higher detergent concentration (1% TX-100) as well as to octylglucoside (1
) and the cytoskeletal components and proteins will not float to the top of the gradient following detergent extraction. Furthermore, we and others have shown previously that M1 protein interacts with cytoskeletal components in influenza virus-infected cells but not in cells expressing either M1 alone or M1 with influenza virus NP (1
Analysis of membrane association of M1 in influenza virus-infected cells (Fig. ) also supports the idea that mature glycoproteins are required for the association of M1 with detergent-resistant membranes and that the newly synthesized M1 can bind to preexisting mature influenza virus glycoproteins, associated with TX-100-resistant lipid rafts in the trans-Golgi membrane and plasma membrane. However, we cannot rule out additional conformational modification of M1 during chase facilitating further M1-glycoprotein interaction. It is possible that the M1-vRNP complex may have further facilitated M1-glycoprotein interactions in influenza virus-infected cells. However, it is clear that M1 can bind to HA or NA in the absence of other influenza virus proteins.
Immunofluorescence analysis by confocal microscopy also supports the interaction of M1 with HA. In influenza virus-infected cells, colocalization of M1 and HA could be seen both at the plasma membrane and in the perinuclear cell cytoplasm but not in the nucleus. Colocalization of HA and M1 was observed both with and without monensin treatment. However, distribution of M1 and HA differed in monensin-treated cells. Almost all HA was present in the perinuclear region after monensin treatment, whereas M1, being more abundant than HA, was not restricted to the perinuclear region but was present throughout the cell. However, the distribution of M1, particularly at the cell periphery, was much less pronounced and distinctly different after monensin treatment due to lack of HA at the plasma membrane (compare Fig. D and J). Essentially similar results were obtained for cells coexpressing HA and M1 from RVVM1- and RVVHA-infected cells. Finally, although biochemical and morphological studies demonstrate interaction of M1 with mature glycoproteins (i.e., glycoproteins present in the mid-Golgi complex and trans-Golgi complex and plasma membrane), we cannot rule out M1-glycoprotein interaction in the cis- or pre-Golgi complex or endoplasmic reticulum.
Coexpression of M1 with heterologous (F) and homologous (HA and NA) glycoproteins showed that homologous glycoproteins were critical for M1 to acquire detergent-resistant membrane association and that heterologous glycoproteins such as Sendai virus F failed to render the membrane-bound M1 detergent resistant (Fig. C). These experiments clearly demonstrated that the interaction of M1 with HA was essential for M1 to become associated with detergent-resistant membranes. Analysis with chimeric constructs between F and HA revealed that both the transmembrane domain and the cytoplasmic tail were involved in interacting with M1. The cytoplasmic tail of HA and the transmembrane domain were independently capable of rendering a fraction of M1 detergent resistant; however, the fraction was less than that obtained with HA or with FHH containing both the transmembrane domain and the cytoplasmic tail of HA (Table ). High-resolution cryoelectron microscopy (6
) and the X-ray crystal structure of the N terminus of M1 (35
) also support the idea that M1 interacts with the inner leaflet of the lipid bilayer and therefore is likely to interact with the COOH half of the transmembrane domain of HA.
The cytoplasmic tails of HA and NA are highly conserved among virus strains. The role of cytoplasmic tails of NA and HA has been investigated using reverse genetics (8
). Since viruses having either tail-minus HA, tail-minus NA, or both tail-minus HA and tail-minus NA could be rescued, it was shown that the cytoplasmic tail of HA and NA individually or together was not an absolute requirement for assembly and particle formation. However, tails of both glycoproteins provided a considerable advantage in efficient budding since the yield of infectious virus in tail-minus mutants was considerably lower and any revertant virus possessing cytoplasmic tail outgrew the mutant viruses (15
). In addition, the influenza virus lacking both tail-minus HA and tail-minus NA exhibited bizarre filamentous morphology (17
). Earlier studies using ts
mutants demonstrated that viral morphogenesis can take place in the absence of either HA or NA (21
), suggesting that there is considerable redundancy in the assembly and budding processes and that only one envelope protein may be sufficient for assembly and budding. However, in none of these experiments was foreign cytoplasmic tail replaced in the transfectant viruses. Furthermore, the role of the transmembrane domain of viral proteins in viral morphogenesis is less clear. HA molecules containing foreign cytoplasmic and foreign transmembrane domains failed to be incorporated into virus particles possessing the wild-type HA (25
), whereas a foreign protein containing the transmembrane domain and cytoplasmic tail of HA was incorporated into virus particles (9
In other viruses, the role of the cytoplasmic tail of the envelope protein in the assembly process and incorporation into virus particles appears to vary greatly. With Sindbis viruses, alteration in the cytoplasmic tail of E2 glycoprotein can prevent particle formation (7
). With vesicular stomatitis virus, the G protein containing a foreign cytoplasmic tail of specific length can be incorporated efficiently (34
), and with rabies virus, budding can take place in the complete absence of spike glycoprotein (23
). Similarly, viruslike particles can be formed and released in the absence of envelope protein in retroviruses including human immunodeficiency virus (4
In conclusion, the data presented here show that a major fraction of influenza virus M1 protein when expressed alone or in virus-infected cells becomes membrane associated immediately after synthesis. Since at this stage M1 protein nonselectively binds to intracellular membranes, the membrane-M1 association is TX-100 detergent soluble. In the presence of homologous viral glycoproteins HA and NA, in either influenza virus-infected cells or cells expressing homologous glycoprotein, M1 interacts with influenza virus glycoproteins and the membrane-M1 interaction becomes TX-100 resistant because of the association of mature HA and NA with lipid rafts enriched in cholesterol and glycosphingolipids. Furthermore, colocalization data reported here indicate that M1 can interact with viral glycoproteins present in the plasma membrane as well as with glycoproteins in transit through the exocytic pathway. M1 interaction with chimeric constructions of glycoproteins demonstrates that both the cytoplasmic tail and the transmembrane domain of influenza virus HA can help membrane-bound M1 to acquire TX-100 resistance, supporting the idea that M1 interacts with both the transmembrane domain and the cytoplasmic tail of HA.