scholarly journals Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus

2005 ◽  
Vol 79 (17) ◽  
pp. 10852-10863 ◽  
Author(s):  
Reyes R. Novoa ◽  
Gloria Calderita ◽  
Pilar Cabezas ◽  
Richard M. Elliott ◽  
Cristina Risco

ABSTRACT Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-β-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.


2022 ◽  
Author(s):  
Kerri L Miazgowicz ◽  
Judith Mary Reyes Ballista ◽  
Marissa D Acciani ◽  
Ariana R Jimenez ◽  
Ryan S Belloli ◽  
...  

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.


2020 ◽  
pp. jbc.RA120.016175
Author(s):  
Bertrand Boson ◽  
Vincent Legros ◽  
Bingjie Zhou ◽  
Eglantine Siret ◽  
Cyrille Mathieu ◽  
...  

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.


2003 ◽  
Vol 77 (2) ◽  
pp. 1368-1381 ◽  
Author(s):  
Iñigo J. Salanueva ◽  
Reyes R. Novoa ◽  
Pilar Cabezas ◽  
Carmen López-Iglesias ◽  
José L. Carrascosa ◽  
...  

ABSTRACT The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.


2015 ◽  
Vol 210 (4) ◽  
pp. 629-646 ◽  
Author(s):  
Jelle Hendrix ◽  
Viola Baumgärtel ◽  
Waldemar Schrimpf ◽  
Sergey Ivanchenko ◽  
Michelle A. Digman ◽  
...  

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.


1988 ◽  
Vol 255 (3) ◽  
pp. 991-998 ◽  
Author(s):  
W McDowell ◽  
A Tlusty ◽  
R Rott ◽  
J N BeMiller ◽  
J A Bohn ◽  
...  

The effects of alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (MMNT) on mannosidases involved in asparagine-linked oligosaccharide processing were investigated. MMNT was found to inhibit the activity of rat liver Golgi alpha-mannosidase I in a concentration-dependent manner (50% inhibition with 0.18 mM-MMNT), whereas rat liver endoplasmic-reticulum alpha-mannosidase appeared to be resistant (less than 5% inhibition at 1 mM-MMNT). Jack-bean alpha-mannosidase was also sensitive to inhibition by MMNT (50% inhibition with 0.32 mM-MMNT). Treatment of influenza-virus-infected chick-embryo cells with 1 mM-MMNT led to a decrease in the formation of complex-type asparagine-linked oligosaccharides and an accumulation of high-mannose-type oligosaccharides with the composition Man8(GlcNAc)2 and Man7(GlcNAc)2 on the viral glycoproteins. The biological activities of influenza-virus haemagglutinin and neuraminidase synthesized in the presence of 1 mM-MMNT remained unchanged, but the virus was less infectious than the control.


2003 ◽  
Vol 77 (20) ◽  
pp. 11105-11113 ◽  
Author(s):  
Colin M. Crump ◽  
Chien-Hui Hung ◽  
Laurel Thomas ◽  
Lei Wan ◽  
Gary Thomas

ABSTRACT The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.


2021 ◽  
Author(s):  
James H Morrison ◽  
Eric Murnane Poeschla

Signal sequences are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus Feline immunodeficiency virus (FIV) contains the longest signal sequence of all eukaryotic, prokaryotic and viral proteins (175 amino acids). The reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here we identify the FIV Env signal sequence (Fess) as the FIV tetherin antagonist. A short deletion in the central portion of Fess had no effect on viral replication in the absence of tetherin but severely impaired virion budding in its presence. Fess is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, it functions by stringently blocking the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane.


Author(s):  
Bertrand Boson ◽  
Vincent Legros ◽  
Bingjie Zhou ◽  
Cyrille Mathieu ◽  
François-Loïc Cosset ◽  
...  

AbstractThe 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, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely β-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.


2021 ◽  
Vol 12 ◽  
Author(s):  
Yury O. Nunez Lopez ◽  
Anna Casu ◽  
Richard E. Pratley

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, is wreaking havoc around the world. Considering that extracellular vesicles (EVs) released from SARS-CoV-2 infected cells might play a role in a viremic phase contributing to disease progression and that standard methods for EV isolation have been reported to co-isolate viral particles, we would like to recommend the use of heightened laboratory safety measures during the isolation of EVs derived from SARS-CoV-2 infected tissue and blood from COVID-19 patients. Research needs to be conducted to better understand the role of EVs in SARS-CoV-2 infectivity, disease progression, and transmission. EV isolation procedures should include approaches for protection from SARS-CoV-2 contamination. We recommend the EV and virology scientific communities develop collaborative projects where relationships between endogenous EVs and potentially lethal enveloped viruses are addressed to better understand the risks and pathobiology involved.


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