scholarly journals Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

2004 ◽  
Vol 85 (5) ◽  
pp. 1181-1189 ◽  
Author(s):  
Sami K. J. Kukkonen ◽  
Antti Vaheri ◽  
Alexander Plyusnin
Keyword(s):  
Matrix Protein ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Expression System ◽  
Peak Level ◽  
Cell Fractionation ◽  
L Protein ◽  
System L ◽  
Infected Cells ◽  
In Cells

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 104 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

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 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 Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture

2008 ◽  
Vol 82 (19) ◽  
pp. 9306-9317 ◽  
Author(s):  
Kyeong-Ok Chang ◽  
David W. George ◽  
John B. Patton ◽  
Kim Y. Green ◽  
Stanislav V. Sosnovtsev
Keyword(s):  
Cell Culture ◽  
Capsid Protein ◽  
Recombinant Plasmid ◽  
Fluorescent Protein ◽  
Feline Calicivirus ◽  
Norwalk Virus ◽  
Human Noroviruses ◽  
Infected Cells ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.

scholarly journals Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli

2000 ◽  
Vol 182 (14) ◽  
pp. 4068-4076 ◽  
Author(s):  
Bradley J. Feilmeier ◽  
Ginger Iseminger ◽  
Diane Schroeder ◽  
Hannali Webber ◽  
Gregory J. Phillips
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Protein Localization ◽  
Cell Fractionation ◽  
Gene Fusions ◽  
Hybrid Protein ◽  
Hybrid Proteins ◽  
Green Fluorescent

ABSTRACT The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfpoptimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by thesec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported β-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

scholarly journals Lettuce necrotic yellows cytorhabdovirus protein localization and interaction map, and comparison with nucleorhabdoviruses

2012 ◽  
Vol 93 (4) ◽  
pp. 906-914 ◽  
Author(s):  
Kathleen M. Martin ◽  
Ralf G. Dietzgen ◽  
Renyuan Wang ◽  
Michael M. Goodin
Keyword(s):  
Protein Interactions ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Bimolecular Fluorescence Complementation ◽  
Cell Periphery ◽  
In Planta ◽  
Interaction Map ◽  
Yellow Dwarf Virus ◽  
P Proteins

Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N–P and M–M interactions are common to all three viruses. Associations unique to only one virus include P–M for LNYV, G–Mv for SYNV and M–Mv, M–G and N–M for PYDV. The cognate N–P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed.

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 Lassa Virus Vaccine Candidate ML29 Generates Truncated Viral RNAs Which Contribute to Interfering Activity and Attenuation

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 214
Author(s):  
Dylan M. Johnson ◽  
Beatrice Cubitt ◽  
Tia L. Pfeffer ◽  
Juan Carlos de la Torre ◽  
Igor S. Lukashevich
Keyword(s):  
Matrix Protein ◽  
Fluorescent Protein ◽  
Vaccine Candidate ◽  
Small Animal ◽  
Lymphocytic Choriomeningitis ◽  
Lassa Virus ◽  
Defective Interfering Particles ◽  
Persistently Infected ◽  
Viral Genomes ◽  
Infected Cells

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1-/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days).

FERRITIN-RED FLUORESCENT PROTEIN FUSION REPORTER FOR MAGNETIC RESONANCE AND OPTICAL IMAGING

2012 ◽  
Vol 24 (04) ◽  
pp. 333-341 ◽  
Author(s):  
Shan-Wen Liu ◽  
Kuan-Hung Cho ◽  
Mei-Ru Chen ◽  
Hsiao-Chi Yu ◽  
Yu-Ying Kao ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Optical Imaging ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Protein Fusion ◽  
Red Fluorescence ◽  
Infected Cells ◽  
Magnetic Resonance Imaging Mri ◽  
High Level ◽  
In Cells

In this report, we generated a ferritin and red fluorescent protein fusion reporter gene that enables the visualization of transgene expression in living animals by magnetic resonance imaging (MRI) and optical imaging. Ferritin heavy chain (FTH) or light chain (FTL) was linked to the N terminus of the monomeric DsRed red fluorescent protein to create FTH-DsRed and FTL-DsRed, MRI-fluorescence dual reporters. Transfection of these dual reporters into cells resulted in increased iron loading and strong red fluorescence in cells. Adenoviral vectors to express FTH-DsRed or FTL-DsRed fusion reporter in infected cells were created, but only the adenovirus expressing FTH-DsRed resulted in a high level of red fluorescence in cells. Delivery and expression of FTH-DsRed in the mouse brain using adenovirus was detected by MRI and fluorescence imaging, revealing a T2shortening effect and an increase of contrast in T2-weighted images at the sites co-localized with strong red fluorescence. While the details of the structure of ferritin-DsRed fusion reporter remains to be solved, this dual reporter is useful for visualizing dynamic processes such as the migration of reporter-transfected stem cells or metastasis of tumors using MRI with the added flexibility of combining optical tools such as fluorescence activated cell sorting and fluorescence microscopy.

scholarly journals Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins

2009 ◽  
Vol 90 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Xiaohong Shi ◽  
Richard M. Elliott
Keyword(s):  
Reverse Genetics ◽  
Immunofluorescent Staining ◽  
Cell Fractionation ◽  
Protein Insertion ◽  
Recombinant Viruses ◽  
L Protein ◽  
Size Growth ◽  
Infected Cells ◽  
L Proteins

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.

scholarly journals Identification of the Rabies Virus Alpha/Beta Interferon Antagonist: Phosphoprotein P Interferes with Phosphorylation of Interferon Regulatory Factor 3

2005 ◽  
Vol 79 (12) ◽  
pp. 7673-7681 ◽  
Author(s):  
Krzysztof Brzózka ◽  
Stefan Finke ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Gene Expression ◽  
Rabies Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Regulatory Factor ◽  
Reading Frame ◽  
Beta Interferon ◽  
P Gene ◽  
Infected Cells ◽  
Alpha Beta

ABSTRACT Rabies virus (RV) of the Rhabdoviridae family grows in alpha/beta interferon (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzózka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-β gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD ΔPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P3, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-β gene expression. In contrast to wt RV, the IFN-inducing SAD ΔPLP caused S386 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor 3 (IRF-3). Phosphorylation of IRF-3 by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-3 by an upstream kinase.

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