scholarly journals Palmitoylations on Murine Coronavirus Spike Proteins Are Essential for Virion Assembly and Infectivity

2006 ◽  
Vol 80 (3) ◽  
pp. 1280-1289 ◽  
Author(s):  
Edward B. Thorp ◽  
Joseph A. Boscarino ◽  
Hillary L. Logan ◽  
Jeffrey T. Goletz ◽  
Thomas M. Gallagher
Keyword(s):  
Acyl Chain ◽  
Molecular Genetic ◽  
Virion Assembly ◽  
M Proteins ◽  
Protein Palmitoylation ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
Intermediate Compartment ◽  
Exocytic Pathway ◽  
S Proteins

ABSTRACT Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.

scholarly journals The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing assembly of virus-like particles

2020 ◽  
pp. jbc.RA120.016175
Author(s):  
Bertrand Boson ◽  
Vincent Legros ◽  
Bingjie Zhou ◽  
Eglantine Siret ◽  
Cyrille Mathieu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Structural Proteins ◽  
Imaging Data ◽  
Virus Like Particles ◽  
Viral Particles ◽  
M Proteins ◽  
Infected Cells ◽  
Intermediate Compartment ◽  
Nucleoprotein N ◽  
Syncytia Formation

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected vs. transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is re-localized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon co-expression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M and N are required for optimal production of virus- like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

scholarly journals Vaccinia Virus 15-Kilodalton (A14L) Protein Is Essential for Assembly and Attachment of Viral Crescents to Virosomes

1998 ◽  
Vol 72 (2) ◽  
pp. 1287-1296 ◽  
Author(s):  
Juan Ramón Rodríguez ◽  
Cristina Risco ◽  
José L. Carrascosa ◽  
Mariano Esteban ◽  
Dolores Rodríguez
Keyword(s):  
Vaccinia Virus ◽  
Electron Microscopic Examination ◽  
Viral Proteins ◽  
Wild Type ◽  
Electron Microscopic ◽  
Small Plaque ◽  
Virion Assembly ◽  
Plaque Phenotype ◽  
Infected Cells ◽  
Intermediate Compartment

ABSTRACT Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacIoperator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-β-d-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interespersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

scholarly journals Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Tu M. Pham ◽  
Si C. Tran ◽  
Yun-Sook Lim ◽  
Soon B. Hwang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Trafficking ◽  
Core Protein ◽  
Cell Types ◽  
Viral Assembly ◽  
Intracellular Membrane ◽  
Virion Assembly ◽  
Hcv Core Protein ◽  
Infected Cells

ABSTRACT Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.

scholarly journals Human Cytomegalovirus Virions Differentially Incorporate Viral and Host Cell RNA during the Assembly Process

2000 ◽  
Vol 74 (19) ◽  
pp. 9078-9082 ◽  
Author(s):  
Astrid E. Greijer ◽  
Chantal A. J. Dekkers ◽  
Jaap M. Middeldorp
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Rna ◽  
Gene Products ◽  
Active Infection ◽  
Virion Assembly ◽  
Viral Genes ◽  
Infected Cells ◽  
Low Levels ◽  
Cellular Dna ◽  
Positive Results

ABSTRACT While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

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 The SARS CoV-1 3a protein disrupts Golgi complex morphology and cargo trafficking

2021 ◽  
Author(s):  
Rex R. Gonzales ◽  
Carolyn E. Machamer
Keyword(s):  
Infectious Bronchitis Virus ◽  
Viral Proteins ◽  
Transfected Cells ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Complex Morphology ◽  
Universal Feature ◽  
Infected Cells ◽  
Intermediate Compartment ◽  
Acidic Compartments

Coronaviruses assemble by budding into the endoplasmic reticulum-Golgi intermediate compartment, but the pathway of egress from infected cells is not well understood. Efficient egress of infectious bronchitis virus (a gamma coronavirus, CoV) requires neutralization of Golgi pH by the envelope (E) protein. This results in reduced rates of cargo traffic and disrupts Golgi morphology, but it protects the spike protein from aberrant proteolysis. The severe acute respiratory syndrome (SARS) CoV-1 E protein does not disrupt the Golgi, however. We show here that in transfected cells, the ORF3a protein of SARS CoV-1 disrupts Golgi morphology, cargo trafficking and luminal pH. Unlike the infectious bronchitis virus E protein, these functions of the SARS CoV-1 3a protein appear to require its viroporin activity. Thus, neutralization of acidic compartments may be a universal feature of CoV infection, although different viral proteins and mechanisms may be used to achieve this outcome.

scholarly journals Inhibiting ACSL1 related ferroptosis restrains MHV-A59 infection

2021 ◽  
Author(s):  
Huawei Xia ◽  
Zeming Zhang ◽  
Fuping You
Keyword(s):  
Hepatitis Virus ◽  
Murine Hepatitis Virus ◽  
Viral Propagation ◽  
Triacsin C ◽  
Representative Model ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
Syncytia Formation ◽  
In Vivo Administration

Murine hepatitis virus strain A59 (MHV-A59) belongs to the β-coronavirus and is considered as a representative model for studying coronavirus infection. MHV-A59 was shown to induce pyroptosis, apoptosis and necroptosis of infected cells, especially the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59, is unknown. Here, we demonstrate inhibiting ferroptosis suppresses MHV-A59 infection. MHV-A59 infection upregulates the expression of Acsl1, a novel ferroptosis inducer. MHV-A59 upregulates Acsl1 expression depending on the NF-kB activation, which is TLR4-independent. Ferroptosis inhibitor inhibits viral propagation, inflammatory cytokines release and MHV-A59 infection induced cell syncytia formation. ACSL1 inhibitor Triacsin C suppresses MHV-A59 infection induced syncytia formation and viral propagation. In vivo administration of liproxstatin-1 ameliorates lung inflammation and tissue injuries caused by MHV-A59 infection. Collectively, these results indicate that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serves as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

scholarly journals An investigation of incorporation of cellular antigens into vaccinia virus particles

2002 ◽  
Vol 83 (10) ◽  
pp. 2347-2359 ◽  
Author(s):  
Oliver Krauss ◽  
Ruth Hollinshead ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Specific Cell ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Membrane Compartments ◽  
Specific Proteins ◽  
Intermediate Compartment ◽  
Low Levels ◽  
Golgi Network ◽  
Exocytic Pathway

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC), cis/medial-Golgi, trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC and cis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.

scholarly journals Porcine Epidemic Diarrhea Virus ORF3 Protein Is Transported through the Exocytic Pathway

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Fusheng Si ◽  
Bingqing Chen ◽  
Xiaoxia Hu ◽  
Ruisong Yu ◽  
Shijuan Dong ◽  
...  
Keyword(s):  
Ion Channel ◽  
Porcine Epidemic Diarrhea Virus ◽  
Intracellular Trafficking ◽  
Expression Vectors ◽  
Orf3 Protein ◽  
Diarrhea Virus ◽  
Terminal Domain ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea ◽  
Exocytic Pathway

ABSTRACT Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein’s subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein’s independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component. IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

Molecular assembly of measles and Nipah virus: specific lipid binding drives conformational change and matrix polymerization

2021 ◽  
Author(s):  
Michael J. Norris ◽  
Monica L. Husby ◽  
William B. Kiosses ◽  
Jieyun Yin ◽  
Linda J. Rennick ◽  
...  
Keyword(s):  
Measles Virus ◽  
Membrane Lipids ◽  
Nipah Virus ◽  
Disease Outbreaks ◽  
Molecular Assembly ◽  
Lipid Binding ◽  
M Protein ◽  
Clear Understanding ◽  
Virion Assembly ◽  
M Proteins

Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. Using X-ray crystallography, electron microscopy, and molecular dynamics we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

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