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At least 19 recordsLinked to original sources
PubMed2003

Nuclear export of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation of viral matrix protein.

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.

Active Transport, Cell Nucleus↗
PubMed1992

Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.

Fluorescence↗
PubMed1997

HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import.

Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.

Amino Acid Sequence↗
PubMed1994

Interaction between tubulin and the viral matrix protein of vesicular stomatitis virus: possible implications in the viral cytopathic effect.

The matrix (M) protein of vesicular stomatitis virus has been shown to induce the rounding of cells. Experiments were performed in order to define the mechanism by which M protein could cause this cytopathic effect (CPE). Immunofluorescence experiments performed on infected cells indicate that cellular rounding coincides with the disruption of the microtubular network. Immunoprecipitation of M protein or tubulin in infected cell extract demonstrates an association of these two proteins in vivo. We show that M protein is capable of interacting in vitro with tubulin in both its polymerized and nonassembled forms. Studies using proteolytically cleaved proteins indicate that this interaction occurs via the highly basic N-terminal domain of M protein and the highly acidic C-terminal region of tubulin. Furthermore, a thermosensitive mutant (tsG33) containing a mutation in the matrix protein gene which is unable to induce CPE at nonpermissive temperature interacts with tubulin with a lower affinity. These results demonstrate that M protein interacts with tubulin in vivo and in vitro and strongly suggest that CPE is caused by this interaction.

Animals↗
PubMed1991

Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import.

Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.

Amantadine↗
PubMed1998

Efficient HIV-1 replication can occur in the absence of the viral matrix protein.

Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells.

Base Sequence↗
PubMed1995

Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein.

OBJECTIVES: To explore the possibility that HIV-1 budding and cellular adhesion molecules co-polarize at cell-to-cell contact sites. To investigate the incorporation of host-cell-derived adhesion molecules into HIV-1. METHODS: The cellular sites involved in HIV-1 budding were examined by transmission electron microscopy. Single and double immunocytochemistry staining was used to evaluate the cellular distribution of the viral matrix protein and adhesion molecules. Quantitative flow cytometry was used to measure the cellular expression of adhesion molecules. An immunocapture technique was used to measure the presence of cell-derived proteins on HIV-1. The captured virus was measured by a p24 antigen assay. The infectivity of virus captured by monoclonal antibodies was tested by measuring the virus antigen yield in supernatants after the addition of sensitive cells. RESULTS: Released and budding HIV-1 was mainly localized at the cell-to-cell contact regions. This feature was consistent with a polarized staining for the virus matrix protein p18 at cell-to-cell contact regions. Intercellular adhesion molecules (ICAM)-1 in HIV-1-infected cells were polarized on both isolated cells and syncytia, co-localizing with HIV-1 matrix protein. HIV-1 incorporated all the adhesion molecules expressed by the host cells, although without quantitative correlation with their cellular expression. CONCLUSIONS: HIV-1 is released at cell-to-cell membrane contact sites. Both ICAM-1 and virus matrix protein co-polarized on isolated cells and syncytia at the sites involved in the recruitment of uninfected cells. The impressive concentration of ICAM at cell sites where most virions are released may account for the acquisition of these membrane proteins by the HIV-1 progeny, and may be important for the cell-mediated spread.

Antigens, CD↗
PubMed2003

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein.

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.

Animals↗
PubMed2007

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.

Amino Acid Sequence↗
PubMed1995

HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator.

The HIV-1 matrix (MA) protein contains two subcellular localization signals with opposing effects. A myristoylated N-terminus governs particle assembly at the plasma membrane, and a nucleophilic motif facilitates import of the viral preintegration complex into the nucleus of nondividing cells. Here, we show that myristoylation acts as the MA dominant targeting signal in HIV-1 producer cells. During virus assembly, a subset of MA is phosphorylated on the C-terminal tyrosine by a virion-associated cellular protein kinase. Tyrosine-phosphorylated MA is then preferentially transported to the nucleus of target cells. An MA tyrosine mutant virus grows normally in dividing cells, but is blocked for nuclear import in terminally differentiated macrophages. MA tyrosine phosphorylation thus reveals the karyophilic properties of this protein within the HIV-1 preintegration complex, thereby playing a critical role for infection of nondividing cells.

Amino Acid Sequence↗
PubMed2005

Oxadiazols: a new class of rationally designed anti-human immunodeficiency virus compounds targeting the nuclear localization signal of the viral matrix protein.

Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

Anti-HIV Agents↗
PubMed2005

Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection.

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4(+) and CD8(+) IFN-gamma(+) T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8(+), but not CD4(+), T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-gamma-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8(+) T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.

Adjuvants, Immunologic↗
PubMed2004

Functional comparison of the two gene products of Thogoto virus segment 6.

The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-beta by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins.

Animals↗
PubMed1985

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells.

B-Lymphocytes↗
PubMed2002

Measles virus matrix protein is not cotransported with the viral glycoproteins but requires virus infection for efficient surface targeting.

As we have shown earlier, the measles virus (MV) glycoproteins H and F are expressed on both, the apical and the basolateral membrane of polarized Madin-Darby canine kidney cells. In contrast to the glycoproteins, we found the viral matrix protein (M) to accumulate selectively at the apical plasma membrane of MV-infected cells. M did not colocalize with the glycoproteins at basolateral membranes of polarized cells indicating an independent surface transport mechanism. Analysis of infected cells treated with monensin supported this view. When H and F were retained in the medial Golgi by monensin treatment, M did not accumulate in this cellular compartment. To elucidate the subcellular transport mechanism of the cytosolic M protein, M was expressed in the absence of other viral proteins. Flotation analysis demonstrated that most of the M protein coflotated in infected or in M-transfected cells with cellular membranes. Thus, the M protein possesses the intrinsic ability to bind to lipid membranes. Unexpectedly, plasmid-encoded M protein was rarely found to accumulate at surface membranes. Although cotransport with the viral glycoproteins was not needed, M transport to the plasma membrane required a component only provided in MV-infected cells.

Animals↗
PubMed1988

Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity.

The peritrophic membrane (PM), which lines the midgut of many insect species, has several functions. In particular, it may serve as a mechanical barrier to invading microorganisms. The protein composition of the PM from healthy and baculovirus-treated Trichoplusia ni (cabbage looper) larvae was analyzed by polyacrylamide gel electrophoresis. A specific interaction took place between baculoviruses and the PM of susceptible T. ni larvae. A 68-kDa glycoprotein of the PM disappeared within 15 min postinoculation with occlusion bodies of either Autographa californica nuclear polyhedrosis virus (AcMNPV) or T. ni nuclear polyhedrosis virus (TnSNPV). In contrast, inoculation of larvae with a T. ni granulosis virus (TnGV) resulted in the disappearance of three distinct major glycoproteins with molecular weights of 253, 194, and 123 kDa. PMs of virus-treated larvae were very fragile compared with those of untreated controls, indicative of a physical/chemical change in their structure. T. ni larval bioassays showed that a factor, present in the TnGV granulin or AcMNPV polyhedrin, enhanced the infectivity of AcMNPV. These data showed that a factor present in the occlusion bodies of three distinct baculoviruses can cause specific biochemical and structural changes in the PM. The biological significance of these observations in relation to increased larval infection is not known at this time.

Animals↗
PubMed1985

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins.

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.

Animals↗
PubMed2000

Crystallization and preliminary X-ray analysis of the matrix protein from Ebola virus.

The matrix protein from Ebola virus is a membrane-associated molecule that plays a role in viral budding. Despite its functional similarity to other viral matrix proteins, it displays no sequence similarity and hence may have a distinct fold. X-ray diffraction quality crystals of the Ebola VP40 matrix protein were grown by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 64.4, b = 91.1, c = 47.9 A, beta = 96.3 degrees. A data set to 1.9 A resolution has been collected using synchrotron radiation. The unit cell contains one molecule of molecular weight 35 kDa per asymmetric unit, with a corresponding volume solvent content of 35%.

Crystallization↗