Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus.

The association of vesicular stomatitis virus proteins with intracellular and plasma membranes was examined by pulse and pulse-chase labeling of virus-infected HeLa cells with [35S]methionine and separation of cell homogenates into three major membrane fractions in discontinuous sucrose gradients. The glycoprotein G was primarily associated with rough endoplasmic reticulum-like membranes after short radioactive pulses (2 to 4 min) but accumulated in the plasma membrane-enriched fraction and the smooth internal membrane fraction with longer pulse or chase periods. The nucleocapsid protein N and the matrix protein M accumulated in the rough endoplasmic reticulum and plasma membrane-like fractions but not in the smooth internal membrane fraction. Only a fraction (35 to 40%) of the viral protein synthesized during a short pulse in the mid-cycle of infection was apparently utilized in released virus. The newly synthesized virus proteins first appeared in released virus in the order: M, N and L, and G.     

Monoclonal antibody #3-9-16 recognizes one of the two isoforms of rabies virus matrix protein that exposes its N-terminus on the virion surface

We investigated behaviors of the rabies virus matrix (M) protein using a monoclonal antibody (mAb), #3-9-16, that recognized a linear epitope located at the N-terminus of the protein. Based on the reactivity with this mAb, M proteins could be divided into at least two isoforms; an ordinary major form (Malpha) whose 3-9-16 epitope is hidden, and an N-terminal-exposed epitope-positive form (Mbeta). The Mbeta protein accounted for about 25-30% of the total M proteins in the virion, while its content in the cell ranged from 10 to 15% of total M protein. Fluorescent antibody (FA) staining showed that the Mbeta antigen distributed in the Golgi area where the colocalized viral glycoprotein antigen was also detected. Mbeta antigen was shown to be exposed on the surface of infected cells by both immunoprecipitation and FA staining with the mAb, whereby the cells might have become sensitive to the mAb-dependent complement-mediated cytolysis. Similarly, the Mbeta antigen was shown to be exposed on the virion surface, and the infectivity of the virus was destroyed by the mAb in the presence of a complement. Together with these results, we think that the M protein molecule takes either of two conformations, one (Mbeta) of which exposes the 3-9-16 epitope located in the N-terminal region of the M protein, that are also exposed on the surface of the virion and infected cells, whereby it might play a certain important role(s) in the virus replication process differently from the other form (Malpha), probably through its intimate association with the Golgi area and/or the cell membrane.     

北京地区两株人偏肺病毒M蛋白基因的原核表达及抗原活性分析

人偏肺病毒(Human metapneumovirus,hMPV)是最近发现的可引起人类呼吸道感染的一种副粘病毒,现被归类于偏肺病毒属(Metapneumovirus),是至今发现的第一个与人类疾病相关的偏肺病毒属成员[1]。目前已经受到世界范围的重视,已有十多个国家报道了不同年龄组人群中hMPV的感染情况。     

用表面等离子体共振技术筛选与禽流感病毒基质蛋白M1相互作用的蛋白

目的：筛选与禽流感病毒基质蛋白M1相互作用的蛋白。方法：表达禽流感病毒基质蛋白M1,经Ni^2＋柱亲和层析纯化,用表面等离子体共振（SPR）技术捕获BHK-21细胞裂解液中与M1相互作用的细胞蛋白,并进行质谱分析。结果：获得了纯度在85%以上的基质蛋白M1,并利用此蛋白捕获到宿主细胞肌球蛋白重链6。结论：肌球蛋白重链6与禽流感病毒基质蛋白M1可能存在体外相互作用。     

Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions.     

利用昆虫杆状病毒表达SARS冠状病毒的刺突蛋白和膜蛋白

SARS冠状病毒是人的严重急性呼吸综合征的病原体。对其他种类冠状病毒的研究结果显示,刺突蛋白(S蛋白)和膜蛋白(M蛋白)是病毒主要的结构蛋白。重组M蛋白和S蛋白可被用来作为抗原检测冠状病毒的感染和制备疫苗。这两个蛋白质分别被克隆并重组到昆虫杆状病毒基因组中,利用重组杆状病毒感染昆虫细胞来表达重组M蛋白和S蛋白,并对M蛋白进行了细胞内定位,融合蛋白的绿色荧光暗示了该蛋白质定位在细胞膜上。     

Interaction of influenza A virus M1 matrix protein with caspases

In this investigation, an ability of influenza A virus M1 matrix protein to bind intracellular caspases, the key enzymes of cell apoptosis, has been examined. Protein–protein binding on polystyrene plates and polyvinyl pyrrolidone membrane was employed for this purpose. Under a comparative study of caspases-3, -6, -7, -8 influenza virus M1 protein specifically bound caspase-8 and weakly bound caspase-7. Using a computer analysis of the N-terminal region of M1 protein, a site similar to the anti-caspase site of baculovirus p35 protein, which inhibits caspases and displays antiapoptotic activity, was identified. These results are in good agreement with the supposition that influenza virus M1 protein is involved in a caspase-8-mediated apoptosis pathway in influenza virus infected cells.     

Impact of Vesicular Stomatitis Virus M Proteins on Different Cellular Functions

Three different matrix (M) proteins termed M1, M2 and M3 have been described in cells infected with vesicular stomatitis virus (VSV). Individual expression of VSV M proteins induces an evident cytopathic effect including cell rounding and detachment, in addition to a partial inhibition of cellular protein synthesis, likely mediated by an indirect mechanism. Analogous to viroporins, M1 promotes the budding of new virus particles; however, this process does not produce an increase in plasma membrane permeability. In contrast to M1, M2 and M3 neither interact with the cellular membrane nor promote the budding of double membrane vesicles at the cell surface. Nonetheless, all three species of M protein interfere with the transport of cellular mRNAs from the nucleus to the cytoplasm and also modulate the redistribution of the splicing factor. The present findings indicate that all three VSV M proteins share some activities that interfere with host cell functions.     

Structural determinants of the interaction between influenza A virus matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95–105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.     

Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus.

Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.     

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.     

Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus.

The membrane-binding affinity of the matrix (M) protein of vesicular stomatitis virus (VSV) was examined by comparing the cellular distribution of wild-type (wt) virus M protein with that of temperature-sensitive (ts) and deletion mutants probed by indirect fluorescent-antibody staining and fractionation of infected or plasmid-transfected CV1 cells. The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive temperature; wt VSV also causes rounding, which prohibits study of M protein distribution by fluorescent-antibody staining. Little or no M protein can be detected in the plasma membrane of cells infected with tsO23 at the nonpermissive temperature, whereas approximately 20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 at the permissive temperature. Cells transfected with a plasmid expressing intact 229-amino-acid wt M protein (M1-229) exhibited cytopathic cell rounding and actin filament dissolution, whereas cells retained normal polygonal morphology and actin filaments when transfected with plasmids expressing M proteins truncated to the first 74 N-terminal amino acids (M1-74) or deleted of the first 50 amino acids (M51-229) or amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were readily detectable in the plasma membrane and cytosol of transfected cells as determined by both fluorescent-antibody staining and cell fractionation, as was the plasmid-expressed intact wt M protein. However, the expressed doubly deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionation, suggesting the presence of two membrane-binding sites spanning the region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in vitro binding assay in which intact M protein and its deletion mutants were reconstituted in high- or low-ionic-strength buffers with synthetic membranes in the form of sonicated unilammelar vesicles. The results of these experiments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membrane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.     

Influenza B virus BM2 protein is transported through the trans-Golgi network as an integral membrane protein

A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

可调控表达甲型流感病毒M2蛋白的哺乳动物细胞系的建立与鉴定

甲型流感病毒M2蛋白是一种具有离子通道功能的跨膜蛋白,其氨基酸序列非常保守,可用于流感通用疫苗的研究。为了构建可调控的稳定表达甲型流感病毒M2蛋白的哺乳动物细胞系,首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传10代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。     

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

Assembly of vesicular stomatitis virus glycoprotein and matrix protein into HeLa cell plasma membranes.

The kinetics of the incorporation of the proteins of vesicular stomatitis virus into the HeLa cell plasma membrane have been studied. The virus M and NS proteins become associated with the plasma membrane very rapidly (< 5 min) while the glycoprotein G shows a lag of about 20 minutes. A similar lag is observed for the incorporation of the G protein into released virus. By pulse-chase experiments the transit time for the G protein from the site of completion to the plasma membrane was also calculated to be about 20 minutes although not all of the G protein could be chased into the plasma membranes.     

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.     

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'- Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.     

Matrix proteins of enveloped viruses: a case study of Influenza A virus M1 protein

Influenza A virus, a member of the Orthomyxoviridae family of enveloped viruses, is one of the human and animal top killers, and its structure and components are therefore extensively studied during the last decades. The most abundant component, M1 matrix protein, forms a matrix layer (scaffold) under the viral lipid envelope, and the functional roles as well as structural peculiarities of the M1 protein are still under heavy debate. Despite multiple attempts of crystallization, no high resolution structure is available for the full length M1 of Influenza A virus. The likely reason for the difficulties lies in the intrinsic disorder of the M1 C-terminal part preventing diffraction quality crystals to be grown. Alternative structural methods including synchrotron small-angle X-ray scattering (SAXS), atomic force microscopy, cryo-electron microscopy/tomography are therefore widely applied to understand the structure of M1, its self-association and interactions with the lipid membrane and the viral nucleocapsid. These methods reveal striking similarities in the behavior of M1 and matrix proteins of other enveloped RNA viruses, with the differences accompanied by the specific features of the viral lifecycles, thus suggesting common interaction principles and, possibly, common evolutional ancestors. The structural information on the Influenza A virus M1 protein obtained to the date strongly suggests that the intrinsic disorder in the C-terminal domain has important functional implications.