The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and Efficiency of Viral Spread In Vitro

In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.     

The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication.

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.     

The Amphipathic Helix of Influenza A Virus M2 Protein Is Required for Filamentous Bud Formation and Scission of Filamentous and Spherical Particles

Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane-proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanines within the amphipathic helix (M2 five-point mutant, or 5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here, we show that any two of these five residues mutated together to alanines result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately 2 logs lower than the wild-type level, and plaque diameter was significantly reduced. When the 5PM and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and the I51A-Y52A A/Udorn/72 and WSN viruses, scission failed, and emerging virus particles exhibited a “beads-on-a-string” morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed, and their abundance at the raft periphery was reduced, suggesting that the M2 amphipathic helix is required for proper localization in the host membrane and that this has implications for budding and scission.     

Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.     

RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.     

Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes

Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated "helix six." This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.     

Quantitation of the glycoprotein spike area on the surface of enveloped viruses

The density of glycoprotein (GP) distribution on the virion surface substantially influences the virus infectivity and pathogenicity. A method to quantitatively determine the area occupied by surface GP spikes was proposed for influenza virus (Flu) strain A/PR/8/34 on the basis of data of tritium bombardment and dynamic light scattering. The latter was used to measure the diameter of intact virions and subviral particles (Flu virions lacking GP spikes after bromelain digestion). Intact virions and subviral particles were bombarded with a hot tritium atom flux, and the specific radioactivity of the matrix M1 protein was analyzed. The tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partly penetrated through the lipid bilayer of the viral envelope, labeling M1, located under the lipid bilayer. The tritium label distribution among different amino acid residues was the same in M1 isolated from subviral particles and M1 isolated from intact virions, demonstrating that the M1 spatial structure remained unchanged during proteolysis of GP spikes. The difference in specific radioactivity between the M1 proteins isolated from intact virions and subviral particles was used to calculate the GP-free portion of the viral surface. Approximating the Flu virion as a sphere, the GP-covered area was estimated at 1.4 × 10⁴ nm², about 40% of the total virion surface. This was consistent with the cryoelectron tomography data published for Flu strain A/X-31. The approach can be applied for other enveloped high pathogenic viruses, such as HIV and the Ebola virus.     

Molecular composition of phenotypically mixed virions formed during mixed infection with influenza viruses A and B

Upon mixed infection of cultured cells by influenza viruses A/WSN/33 and B/Lee/40 the produced virions contain in their envelopes either hemagglutinin B/Lee/40, or hemagglutinins of both viruses, depending on their concentration ratio during infection. In the first case the population contains RNA segments and nucleoproteins (NP) of both viruses, in the second-exclusively RNA and NP of virus A/WSN/33. Results of immunoprecipitation with monoclonal antibodies to protein of virus A/WSN/33 with further analysis of immunoprecipitates by electrophoresis in polyacrylamide gels did not reveal the presence of virus ribonucleoproteins, containing NP of both viruses. The data obtained demonstrate the high of specificity of protein-protein recognition during reassembly of virion inner structures.     

Role of hemagglutinin cleavage and expression of M1 protein in replication of A/WS/33, A/PR/8/34, and WSN influenza viruses in mouse brain.

The combined presence of WSN gene segments 6 (neuraminidase), 7 (M1 and M2), and 8 (NS1 and NS2) in reassortants of WSN with A/Aichi/2/68 (H3N2) has been found by others to be necessary for full expression of neurovirulence in mice. We are examining the expression of the analogous three gene segments in brains of mice after intracerebral infection with non-neuroadapted strains A/WS/33 (WS) (from which WSN was derived) and A/PR/8/34 (PR8). Our aim is to determine possible mechanisms by which one or more of the five gene products may restrict replication of these strains in mouse brain cells to a single cycle, yielding noninfectious hemagglutinating particles (incomplete growth cycle). We found that minority subsets of such particles did produce plaques, provided they were activated by trypsin (analogous to other abortive systems producing virions with uncleaved HA), a step obviated for some WSN virions by indirect promotion of hemagglutinin cleavage by the neuraminidase of that strain. The percentage of such potentially infectious virions, relative to total hemagglutinating particles, was significantly lower in WS- or PR8-infected than in WSN-infected brains, suggesting possible defects in synthesis or function of M1 protein in the former. Cells in immunostained sections and appropriate bands in Western blots (immunoblots) of viral proteins electrophoretically separated from lysates of PR8-infected brains reacted with antibody to nucleoprotein but not to M1 protein. Either method revealed the presence of both proteins in WSN-infected brains. In contrast, Western blot analyses of particles concentrated from PR8-, WS-, or WSN-infected brains by hemadsorption, elution, and pelleting did reveal NP and M1 bands with comparable relative peroxidase-antiperoxidase staining intensities. The findings suggest that availability of M1 protein is a factor influencing the extent or rate of assembly of potentially infectious (i.e., trypsin-activated) progeny virions in mouse brains and that in this respect the two non-neurovirulent strains differ from WSN quantitatively rather than qualitatively.     

Growth control of influenza A virus by M1 protein: analysis of transfectant viruses carrying the chimeric M gene.

Analysis of fast-growing reassortants (AWM viruses) of influenza A virus produced by mixed infection with a fast-growing WSN strain and a slowly growing Aichi strain indicated that the M gene plays a role in the regulation of virus growth rate at an early step of infection (J. Yasuda, T. Toyoda, M. Nakayama, and A. Ishihama, Arch. Virol. 133:283-294, 1993). To determine which of the two M gene products, M1 or M2, is responsible for the growth rate control, one recombinant WSN virus (CWA) clone possessing a chimeric M gene (WSN M1-Aichi M2) was generated by using an improved reverse genetics and transfection system. The recombinant CWA virus retained the phenotype of both large plaque formation and early onset of virus growth. This indicates that the WSN M1 protein is responsible for rapid virus growth.     

Filamentous Influenza Virus Enters Cells via Macropinocytosis

Influenza virus is pleiomorphic, producing both spherical (100-nm-diameter) and filamentous (100-nm by 20-μm) virions. While the spherical virions are known to enter host cells through exploitation of clathrin-mediated endocytosis, the entry pathway for filamentous virions has not been determined, though the existence of an alternative, non-clathrin-, non-caveolin-mediated entry pathway for influenza virus has been known for many years. In this study, we confirm recent results showing that influenza virus utilizes macropinocytosis as an alternate entry pathway. Furthermore, we find that filamentous influenza viruses use macropinocytosis as the primary entry mechanism. Virions enter cells as intact filaments within macropinosomes and are trafficked to the acidic late-endosomal compartment. Low pH triggers a conformational change in the M2 ion channel protein, altering membrane curvature and leading to a fragmentation of the filamentous virions. This fragmentation may enable more-efficient fusion between the viral and endosomal membranes.     

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.     

Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells.

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.     

Temperature-sensitive BC mutants of simian virus 40: block in virion assembly and accumulation of capsid-chromatin complexes.

We examined the morphology, protein composition, and stability of the nucleoprotein complexes assembled in cells infected with simian virus 40 mutants belonging to the BC complementation group (tsBC11, tsBC208, tsBC214, tsB216, tsBC217, tsBC248, tsBC223, and tsBC274). We found that the 220S virions were not assembled in tsBC-infected cells under restrictive conditions. This block in assembly resulted in the accumulation of 75S chromatin in tsBC11-infected cells, as previously observed by Garber et al. (E.A. Garber, M.M. Seidman, and A.J. Levine, Virology 107:389-401, 1980). In cells infected with any other mutant listed above, the block in assembly resulted in the accumulation of 75S chromatin as well as nucleoprotein complexes sedimenting from 90 to 140S. Biochemical analysis revealed that these latter complexes contained the capsid proteins in addition to simian virus 40 DNA and the cellular core histones. Electron microscopic analysis clearly showed the association of the capsid proteins with the viral chromatin. Our results suggest that these proteins interact with simian virus 40 chromatin in the course of virion maturation and may thus play an active role in controlling simian virus 40 functions.     

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.     

A mutation on influenza C virus M1 protein affects virion morphology by altering the membrane affinity of the protein

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae. We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 x 10(3) 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (10(6)/ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala-->Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology.     

Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.