Replication of a Nipah Virus Encoding a Nuclear-Retained Matrix Protein

2019 ◽  
Vol 221 (Supplement_4) ◽  
pp. S389-S394 ◽  
Author(s):  
Marc Ringel ◽  
Laura Behner ◽  
Anja Heiner ◽  
Lucie Sauerhering ◽  
Andrea Maisner
Keyword(s):  
Plasma Membrane ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Nipah Virus ◽  
Nuclear Export Signal ◽  
Cell Types ◽  
Surface Transport ◽  
M Proteins ◽  
Infected Cells

Abstract Nipah virus (NiV) matrix protein (NiV M) plays a major role in virus assembly. It undergoes nuclear transit before accumulating at the plasma membrane and recruiting nucleocapsids to the budding sites. Because nuclear NiV M cannot be detected in all cell types, we wondered whether it can reach the cell surface by bypassing the nucleus. Using an M mutant with a defective nuclear export signal (MNESmut), however, we revealed that the nuclear import of M is ubiquitous, because MNESmut was retained in the nuclei of all cell types tested. Because a functional nuclear transit is a general prerequisite for M surface transport, we wanted to characterize the effect of nuclear-retained M protein in a full viral context and generated a recombinant NiV-MNESmut. Mutant NiV-MNESmut caused increased cell-cell fusion and produced lower virus titers. As expected for an assembly defective NiV, perinuclear inclusions (IBperi) were formed, but inclusions at the plasma membrane (IBPM), which probably represent the viral assembly platforms, were not found. It is interesting to note that the transport-defective MNESmut was recruited to IBperi. This probably prevents overaccumulation of nonfunctional M proteins in the cytoplasm and nuclei of NiV-infected cells and thus provides first evidence that IBperi are functionally relevant aggresome-like compartments.

scholarly journals The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

2009 ◽  
Vol 83 (11) ◽  
pp. 5353-5362 ◽  
Author(s):  
Reena Ghildyal ◽  
Adeline Ho ◽  
Manisha Dias ◽  
Lydia Soegiyono ◽  
Phillip G. Bardin ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Nuclear Export Signal ◽  
M Protein ◽  
Nuclear Accumulation ◽  
Infected Cells ◽  
Syncytial Virus ◽  
In Cells

ABSTRACT The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.

scholarly journals Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization

2006 ◽  
Vol 80 (24) ◽  
pp. 12070-12078 ◽  
Author(s):  
Michael J. Ciancanelli ◽  
Christopher F. Basler
Keyword(s):  
Matrix Protein ◽  
Ebola Virus ◽  
Equine Infectious Anemia Virus ◽  
Nipah Virus ◽  
Gag Protein ◽  
Late Domain ◽  
M Proteins ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.

scholarly journals C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

2016 ◽  
Vol 90 (7) ◽  
pp. 3650-3660 ◽  
Author(s):  
Greeshma Ray ◽  
Phuong Tieu Schmitt ◽  
Anthony P. Schmitt
Keyword(s):  
Nucleocapsid Protein ◽  
Matrix Protein ◽  
Nipah Virus ◽  
Mumps Virus ◽  
Foreign Protein ◽  
Efficient Production ◽  
Nucleocapsid Proteins ◽  
Virus Like Particles ◽  
Ribonucleoprotein Complexes ◽  
M Proteins

ABSTRACTParamyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein,Renillaluciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities.IMPORTANCEParamyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools.

scholarly journals Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

scholarly journals Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

2004 ◽  
Vol 78 (10) ◽  
pp. 5358-5367 ◽  
Author(s):  
Jason J. Rodriguez ◽  
Cristian D. Cruz ◽  
Curt M. Horvath
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Nipah Virus ◽  
Nuclear Export Signal ◽  
Hendra Virus ◽  
Nuclear Accumulation ◽  
V Protein ◽  
Binding Domains ◽  
Export Signal

ABSTRACT The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

scholarly journals Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Melissa Batonick ◽  
Gail W. Wertz
Keyword(s):  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
G Proteins ◽  
Matrix Protein ◽  
Protein Localization ◽  
Viral Proteins ◽  
Human Respiratory Syncytial Virus ◽  
The Other ◽  
Infected Cells ◽  
Syncytial Virus

Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.

scholarly journals Reply to “Nuclear Export Signal and Immunodominant CD8+T Cell Epitope in Influenza A Virus Matrix Protein 1”

2012 ◽  
Vol 86 (18) ◽  
pp. 10259-10260
Author(s):  
Shuai Cao ◽  
Yi Shi ◽  
Shuguang Tan ◽  
Hao Song ◽  
George F. Gao ◽  
...  
Keyword(s):  
T Cell ◽  
Influenza A Virus ◽  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Nuclear Export Signal ◽  
Cell Epitope ◽  
Cd8 T Cell ◽  
T Cell Epitope ◽  
Export Signal

scholarly journals Importin-β Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid

2006 ◽  
Vol 80 (4) ◽  
pp. 1798-1806 ◽  
Author(s):  
Kristin L. Butterfield-Gerson ◽  
Lisa Z. Scheifele ◽  
Eileen P. Ryan ◽  
Anita K. Hopper ◽  
Leslie J. Parent
Keyword(s):  
Nuclear Export ◽  
Virus Assembly ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Targeting ◽  
Nuclear Entry ◽  
Gag Protein ◽  
Gag Polyprotein ◽  
In The Wild ◽  
Importin Α

ABSTRACT The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-β family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-α/β (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

scholarly journals Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane

2018 ◽  
Vol 92 (10) ◽  
Author(s):  
Ritsuko Koga ◽  
Marie Kubota ◽  
Takao Hashiguchi ◽  
Yusuke Yanagi ◽  
Shinji Ohno
Keyword(s):  
Plasma Membrane ◽  
Measles Virus ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Annexin A2 ◽  
Host Protein ◽  
Host Cells ◽  
M Protein ◽  
Dependent Manner ◽  
Inner Surface

ABSTRACTAnnexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca2+-dependent manner. Annexin A2 (AnxA2), a member of this family, has been implicated in a variety of cellular functions, including the organization of membrane domains, vesicular trafficking, and cell-cell adhesion. AnxA2 generally forms a heterotetrameric complex with a small Ca2+-binding protein, S100A10. Measles virus (MV), a member of the familyParamyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells without affecting its entry and viral RNA production. In MV-infected, AnxA2 knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10 and was colocalized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6 to 10), thereby aiding in MV assembly.IMPORTANCEMV is an important human pathogen, still claiming ∼100,000 lives per year despite the presence of effective vaccines, and it causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV M protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of antiviral agents.

Sign in / Sign up

Export Citation Format

Share Document