Influenza virus enters its host cell by receptor-mediated endocytosis followed by acid-activated membrane fusion in endosomes. The viral ribonucleoprotein particles (vRNPs) delivered into the cytosol then dissociate from the matrix protein, M1, and from each other, after which they are individually imported into the nucleus via the nuclear pores. For some time, it has been believed that the low pH in endosomes may, in some way, trigger the capsid disassembly events necessary for nuclear transport. This report provides direct evidence that the association of M1 with vRNPs is sensitive to mildly acidic pH within the infected cell. Recombinant M1, expressed in cultured cells, was found to associate with vRNPs and inhibit their nuclear import. Brief acidification of the cytosolic compartment eliminated the interfering activity and allowed the incoming vRNPs to enter the nucleus. Newly assembled progeny M1-vRNP complexes in the cytosol of infected cells were also dissociated by brief acidification. Acidic pH was thus found to serve as a switch that allowed M1 to carry out its multiple functions in the uncoating, nuclear transport, and assembly of vRNPs.
The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32 P i into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.Influenza virus is a negative-sense RNA virus which replicates in the cell nucleus. Infection begins with the virus binding to the plasma membrane, followed by receptor-mediated endocytosis and a low-pH-dependent fusion step in the late endosome (20,37). This releases the viral ribonucleoproteins (vRNPs), together with the matrix protein (M1), into the cytoplasm. We have previously shown that the dissociation of M1 from the vRNPs allows subsequent import of the vRNPs into the nucleus (11,18).The first viral proteins to be synthesized are the polypeptides of the polymerase complex (PA, PB1, and PB2), the nonstructural protein NS-1, and the nucleoprotein (NP), with synthesis commencing within 1 to 2 h of infection (17). These proteins are all imported into the nucleus shortly after their synthesis. Later in infection, the remainder of the virus proteins, hemagglutinin (HA) and neuraminidase (NA), the nonstructural protein NS-2, and the M1 and M2 proteins, are synthesized. HA, NA, and M2 are synthesized at the endoplasmic reticulum and transported to the plasma membrane, where they are available for virus budding. The M1 protein is distributed to the nucleus shortly after its synthesis (2, 24).The newly synthesized NP associates with the negative-sense viral RNA in the nucleus, thereby forming the vRNPs, which remain in the nucleus until the onset of M1 synthesis. After its import into the nucleus, M1 protein acts in an, as yet, unknown manner to promote export of the vRNPs from the nucleus to the cytoplasm (18). The vRNPs are further transported to the plasma membrane, where additional M1 molecules are inv...
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