scholarly journals Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

2006 ◽  
Vol 80 (16) ◽  
pp. 8089-8099 ◽  
Author(s):  
Xiaohong Shi ◽  
Alain Kohl ◽  
Vincent H. J. Léonard ◽  
Ping Li ◽  
Angela McLees ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Nonstructural Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Terminal Region ◽  
Coding Region ◽  
Hydrophobic Domain

ABSTRACT The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

scholarly journals Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Benjamin Brennan ◽  
Veronica V. Rezelj ◽  
Richard M. Elliott
Keyword(s):  
Virus Infection ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transcription Termination ◽  
Severe Disease ◽  
Open Reading Frame ◽  
Coding Region ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

scholarly journals Movement of Bax from the Cytosol to Mitochondria during Apoptosis

1997 ◽  
Vol 139 (5) ◽  
pp. 1281-1292 ◽  
Author(s):  
Keith G. Wolter ◽  
Yi-Te Hsu ◽  
Carolyn L. Smith ◽  
Amotz Nechushtan ◽  
Xu-Guang Xi ◽  
...  
Keyword(s):  
Soluble Protein ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Mitochondrial Marker ◽  
Nuclear Condensation ◽  
Hydrophobic Domain ◽  
Induction Of Apoptosis ◽  
Green Fluorescent ◽  
Promote Cell Death

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP–Bcl-2 and GFP–Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP–Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP–Bax is a soluble protein, in contrast to organelle-bound GFP–Bcl-2. The diffuse localization of GFP–Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP–Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP–Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP– Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

scholarly journals Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.

1990 ◽  
Vol 110 (4) ◽  
pp. 999-1011 ◽  
Author(s):  
R G Paterson ◽  
R A Lamb
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Protein Fusion ◽  
Native Form ◽  
Hydrophobic Domain ◽  
External Domain ◽  
Signal Anchor

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.

scholarly journals Reverse Genetics System for Uukuniemi Virus (Bunyaviridae): RNA Polymerase I-Catalyzed Expression of Chimeric Viral RNAs

2001 ◽  
Vol 75 (4) ◽  
pp. 1643-1655 ◽  
Author(s):  
Ramon Flick ◽  
Ralf F. Pettersson
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Rna Polymerase I ◽  
Gfp Expression ◽  
Pol I ◽  
Green Fluorescent ◽  
Polymerase I ◽  
Expression Plasmids

ABSTRACT We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of theBunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5′- and 3′-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

scholarly journals Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4317-4328 ◽  
Author(s):  
M A Williams ◽  
R A Lamb
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Terminal Region ◽  
Influenza B Virus ◽  
Influenza B ◽  
Directed Mutagenesis ◽  
Cell Fractionation ◽  
B Virus

The membrane orientation of the NB protein of influenza B virus, a small (Mr, approximately 18,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.

scholarly journals Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-Tagged Gc Glycoprotein

2010 ◽  
Vol 84 (17) ◽  
pp. 8460-8469 ◽  
Author(s):  
Xiaohong Shi ◽  
Joël T. van Mierlo ◽  
Andrew French ◽  
Richard M. Elliott
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Virus Assembly ◽  
Morphological Changes ◽  
Viral Attachment ◽  
Virus Attachment ◽  
Recombinant Viruses ◽  
Mammalian Cell Lines ◽  
Golgi Compartment

ABSTRACT The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also of the Orthobunyavirus genus, are encoded by the medium (M) RNA genome segment and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the endoplasmic reticulum (ER) and transit to the Golgi compartment for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (X. Shi, J. Goli, G. Clark, K. Brauburger, and R. M. Elliott, J. Gen. Virol. 90:2483-2492, 2009.). In this study, the coding sequence for a fluorescent protein, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, was fused to the N terminus of truncated Gc, and two recombinant BUNVs (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages, whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP, resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi compartment at later stages of infection. The fluorescent protein-tagged viruses will be valuable reagents for live-cell imaging studies to investigate virus entry, budding, and morphogenesis in real time.

scholarly journals Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum.

1994 ◽  
Vol 125 (4) ◽  
pp. 803-815 ◽  
Author(s):  
W R Skach ◽  
L B Shi ◽  
M C Calayag ◽  
A Frigeri ◽  
V R Lingappa ◽  
...  
Keyword(s):  
Structural Model ◽  
Signal Sequence ◽  
Water Channel ◽  
Integral Membrane Protein ◽  
Coding Region ◽  
Hydrophobic Region ◽  
Transmembrane Topology ◽  
Nascent Chain ◽  
Membrane Spanning ◽  
Er Membrane

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.

scholarly journals Correct Promoter Control Is Needed for Trafficking of the Ring-Infected Erythrocyte Surface Antigen to the Host Cytosol in Transfected Malaria Parasites

2004 ◽  
Vol 72 (10) ◽  
pp. 6095-6105 ◽  
Author(s):  
Melanie Rug ◽  
Mark E. Wickham ◽  
Michael Foley ◽  
Alan F. Cowman ◽  
Leann Tilley
Keyword(s):  
Surface Antigen ◽  
Transgene Expression ◽  
Infected Erythrocyte ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Parasitophorous Vacuole ◽  
Chimeric Protein ◽  
Terminal Region ◽  
Signal Sequences ◽  
Erythrocyte Surface

ABSTRACT Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependant on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.

scholarly journals Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

2007 ◽  
Vol 81 (18) ◽  
pp. 10151-10160 ◽  
Author(s):  
Xiaohong Shi ◽  
Alain Kohl ◽  
Ping Li ◽  
Richard M. Elliott
Keyword(s):  
Reverse Genetics ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Glycoprotein Precursor

ABSTRACT The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

scholarly journals Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

2009 ◽  
Vol 84 (4) ◽  
pp. 1666-1673 ◽  
Author(s):  
Martina Kopp ◽  
Catherine L. Murray ◽  
Christopher T. Jones ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Virus Production ◽  
Terminal Region ◽  
Dual Processing ◽  
Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

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