Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus

Abstract

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...

Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells

“…Taken together, these results would support the notion that to acquire TX-100 insolubility, M1 needs vRNP in the cytoplasm. Unlike ts56, a ts mutant of M1 (ts51) exhibited similar TX-100 insolubility to the wild-type M1 (data not shown), except that less M1 was present in the cytoplasm after the chase, as expected (55,73).…”

“…Influenza virus M1 and NP are present in both the nucleus and cytoplasm (6,50) and appear to possess nuclear targeting signals (75). However, the nuclear and cytoplasmic localizations of M1 and NP appears to depend on the time of the infectious cycle, i.e., early versus late (55,72), and modifications such as phosphorylation (73). Since in this study we wanted to investigate the interactions of cytoplasmic M1 and NP with host components, we examined the cytoplasmic and nuclear distribution of M1 and NP in virus-infected cells.…”

Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

“…The influenza virus M 1 protein associates with RNPs in the nucleus and is involved with the NS 2 protein in the export of the RNPs from the nucleus (37,44,63). A fraction of the pool of M 1 protein also associates with cellular membrane fractions (1,9,15,17,26,67), and it is generally believed that M 1 protein associates with the cytoplasmic tails of HA and NA.…”

Rna Transport

“…Export of vRNPs is dependent on nuclear-localized M1 protein (Fraser 1967, Martin & Helenius 1991. The mechanism by which M1 functions has not yet been characterized, although studies of the transport properties of vRNPs of a virus bearing a temperaturesensitive transport mutation in M1 have been informative (Whittaker et al 1995, Ye et al 1995. At the nonpermissive temperature, most of the mutant M1 protein appears to be restricted to the nucleus, and the vRNPs are exported without binding detectable M1 protein.…”