Defective Viral Genomes Alter How Sendai Virus Interacts with Cellular Trafficking Machinery, Leading to Heterogeneity in the Production of Viral Particles among Infected Cells

ABSTRACTDefective viral genomes (DVGs) generated during RNA virus replication determine infection outcome by triggering innate immunity, diminishing virulence, and, in many cases, facilitating the establishment of persistent infections. Despite their critical role during virus-host interactions, the mechanisms regulating the production and propagation of DVGs are poorly understood. Visualization of viral genomes using RNA fluorescentin situhybridization revealed a striking difference in the intracellular localization of DVGs and full-length viral genomes during infections with the paramyxovirus Sendai virus. In cells enriched in full-length virus, viral genomes clustered in a perinuclear region and associated with cellular trafficking machinery, including microtubules and the GTPase Rab11a. However, in cells enriched in DVGs, defective genomes distributed diffusely throughout the cytoplasm and failed to interact with this cellular machinery. Consequently, cells enriched in full-length genomes produced both DVG- and full-length-genome-containing viral particles, while DVG-high cells poorly produced viral particles yet strongly stimulated antiviral immunity. These findings reveal the selective production of both standard and DVG-containing particles by a subpopulation of infected cells that can be differentiated by the intracellular localization of DVGs. This study highlights the importance of considering this functional heterogeneity in analyses of virus-host interactions during infection.IMPORTANCEDefective viral genomes (DVGs) generated during Sendai virus infections accumulate in the cytoplasm of some infected cells and stimulate antiviral immunity and cell survival. DVGs are packaged and released as defective particles and have a significant impact on infection outcome. We show that the subpopulation of DVG-high cells poorly engages the virus packaging and budding machinery and do not effectively produce viral particles. In contrast, cells enriched in full-length genomes are the primary producers of both standard and defective viral particles during infection. This study demonstrates heterogeneity in the molecular interactions occurring within infected cells and highlights distinct functional roles for cells as either initiators of immunity or producers and perpetuators of viral particles depending on their content of viral genomes and their intracellular localization.

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Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

Journal of Virology ◽

10.1128/jvi.73.9.7126-7131.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7126-7131 ◽

Cited By ~ 49

Author(s):

Catherine E. Patterson ◽

Thomas Shenk

Keyword(s):

Human Cytomegalovirus ◽

Half Life ◽

Cultured Cells ◽

Intracellular Localization ◽

State Level ◽

Specific Variation ◽

Infected Cells ◽

Ad169 Strain ◽

Expression Plasmids ◽

In Cells

ABSTRACT Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.

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Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

Proceedings ◽

10.3390/proceedings2020050120 ◽

2020 ◽

Vol 50 (1) ◽

pp. 120

Author(s):

Juan-Vicente Bou ◽

Ron Geller ◽

Rafael Sanjuán

Keyword(s):

Genetic Variants ◽

Social Evolution ◽

Viral Population ◽

Multiplicity Of Infection ◽

Defective Interfering Particles ◽

Viral Genomes ◽

Viral Particles ◽

A Cell ◽

Infected Cells ◽

Population Sequence

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion.

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The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious

Viruses ◽

10.3390/v13102034 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2034

Author(s):

Shan-Ying Wu ◽

Yu-Lun Chen ◽

Ying-Ray Lee ◽

Chiou-Feng Lin ◽

Sheng-Hui Lan ◽

...

Keyword(s):

Dengue Virus ◽

Full Length ◽

Genomic Rnas ◽

Double Membrane ◽

Transmission Electron ◽

Viral Particles ◽

Autophagic Degradation ◽

Infected Cells ◽

Intracellular Vesicles ◽

Secretory Autophagy

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.

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Cleavage of IPS-1 in Cells Infected with Human Rhinovirus

Journal of Virology ◽

10.1128/jvi.01490-09 ◽

2009 ◽

Vol 83 (22) ◽

pp. 11581-11587 ◽

Cited By ~ 56

Author(s):

Jennifer Drahos ◽

Vincent R. Racaniello

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Sendai Virus ◽

Innate Immune ◽

Human Rhinovirus ◽

Type I ◽

Human Pathogens ◽

Infected Cells ◽

In Cells

ABSTRACT Rhinoviruses are prevalent human pathogens that are associated with life-threatening acute asthma exacerbations. The innate immune response to rhinovirus infection, which may play an important role in virus-induced asthma induction, has not been comprehensively investigated. We examined the innate immune response in cells infected with human rhinovirus 1a (HRV1a). Beta interferon (IFN-β) mRNA was induced in HRV1a-infected cells at levels significantly lower than in cells infected with Sendai virus. To understand the basis for this observation, we determined whether components of the pathway leading to IFN-β induction were altered during infection. Dimerization of the transcription factor IRF-3, which is required for synthesis of IFN-β mRNA, is not observed in cells infected with HRV1a. Beginning at 7 h postinfection, IPS-1, a protein that is essential for cytosolic sensing of viral RNA, is degraded in HRV1a-infected cells. Induction of apoptosis by puromycin led to the cleavage of IPS-1, but treatment of HRV1a-infected cells with the pan-caspase inhibitor, zVAD, did not block cleavage of IPS-1. IPS-1 is cleaved in vitro by caspase-3 and by the picornaviral proteinases 2Apro and 3Cpro. Expression of HRV1a and polioviral 2Apro and 3Cpro led to degradation of IPS-1 in cells. These results suggest that IPS-1 is cleaved during HRV1a infection by three different proteases. Cleavage of IPS-1 may be a mechanism for evasion of the type I IFN response, leading to a more robust infection.

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The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

10.1101/2020.04.23.058883 ◽

2020 ◽

Author(s):

Emmanuelle Genoyer ◽

Katarzyna Kulej ◽

Chuan Tien Hung ◽

Patricia A. Thibault ◽

Kristopher Azarm ◽

...

Keyword(s):

Sendai Virus ◽

Initial Step ◽

Viral Proteins ◽

Viral Polymerase ◽

Recycling Endosomes ◽

Viral Genomes ◽

Parainfluenza Viruses ◽

Infected Cells ◽

Human Parainfluenza Viruses

ABSTRACTParamyxoviruses are negative sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here we take advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examine the role of matrix protein and nucleoprotein and determine that they are not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative protein abundance and localization in DVG- and FL-high cells, we identify viral polymerase complex components L and, specifically, its cofactor C proteins as interactors with Rab11a. We find that accumulation of these proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells which remain in replication phases.IMPORTANCEParamyxoviruses are a family of viruses that include a number of pathogens with significant burdens on human health. Particularly, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or direct acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and coopt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here we use the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We find that viral polymerase components large protein L and accessory C proteins are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

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Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence

10.1101/120766 ◽

2017 ◽

Author(s):

Jie Xu ◽

Yan Sun ◽

Yize Li ◽

Gordon Ruthel ◽

Susan R. Weiss ◽

...

Keyword(s):

Cell Fate ◽

Sendai Virus ◽

Rna Virus ◽

Viral Persistence ◽

Virus Infections ◽

Viral Genomes ◽

Persistent Infections ◽

Infected Cells ◽

Syncytial Virus ◽

Survival Effect

ABSTRACTReplication defective viral genomes (DVGs) generated during virus replication are the primary triggers of antiviral immunity in many RNA virus infections. However, DVGs can also facilitate viral persistence. Why and how these two opposing functions of DVGs are achieved remain unknown. Here we report that during Sendai and respiratory syncytial virus infections DVGs selectively protect a subpopulation of cells from death and promote the establishment of persistent infections. We find that during Sendai virus infection this phenotype results from DVGs stimulating a MAVS-mediated TNF response that drives apoptosis of highly infected cells while extending the survival of cells enriched in DVGs. The pro-survival effect of TNF depends on the activity of the TNFR2/TRAF1 pathway that is regulated by MAVS signaling. These results identify TNF as a pivotal factor in determining cell fate during a viral infection and delineate a MAVS/TNFR2-mediated mechanism that drives the persistence of otherwise acute viruses.

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Live Visualization of Hemagglutinin Dynamics during Infection by Using a Novel Reporter Influenza A Virus

Viruses ◽

10.3390/v12060687 ◽

2020 ◽

Vol 12 (6) ◽

pp. 687

Author(s):

Luiz Gustavo dos Anjos Borges ◽

Giuseppe Pisanelli ◽

Oyahida Khatun ◽

Adolfo García-Sastre ◽

Shashank Tripathi

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Immunofluorescence Assay ◽

Confocal Imaging ◽

Host Interactions ◽

Chemical Inhibitors ◽

Versatile Tool ◽

Infected Cells ◽

Ha Protein ◽

In Cells

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing.

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The Viral Polymerase Complex Mediates the Interaction of Viral Ribonucleoprotein Complexes with Recycling Endosomes during Sendai Virus Assembly

mBio ◽

10.1128/mbio.02028-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Emmanuelle Genoyer ◽

Katarzyna Kulej ◽

Chuan Tien Hung ◽

Patricia A. Thibault ◽

Kristopher Azarm ◽

...

Keyword(s):

Sendai Virus ◽

Initial Step ◽

Viral Proteins ◽

Viral Polymerase ◽

Recycling Endosomes ◽

Viral Genomes ◽

Ribonucleoprotein Complexes ◽

Parainfluenza Viruses ◽

Infected Cells ◽

Human Parainfluenza Viruses

ABSTRACT Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here, we used the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) (DVG-high SeV) generates heterogenous populations of cells. Cells enriched in full-length (FL) virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here, we took advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examined the roles of matrix protein and nucleoprotein and determined that their presence is not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative analyses of protein abundance and localization in DVG-high and FL-virus-high (FL-high) cells, we identified viral polymerase complex component protein L and, specifically, its cofactor C as interactors with Rab11a. We found that accumulation of L and C proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells containing viruses that remain in replication phases. IMPORTANCE Paramyxoviruses are members of a family of viruses that include a number of pathogens imposing significant burdens on human health. In particular, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or directly acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and co-opt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here, we used the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We found that the viral polymerase components large protein L and accessory protein C are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

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An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging

10.1101/676031 ◽

2019 ◽

Cited By ~ 1

Author(s):

Camila Pereira-Montecinos ◽

Daniela Toro-Ascuy ◽

Cecilia Rojas-Fuentes ◽

Sebastián Riquelme-Barrios ◽

Bárbara Rojas-Araya ◽

...

Keyword(s):

Full Length ◽

Genomic Rna ◽

Rna Packaging ◽

Gag Protein ◽

Rna Molecules ◽

Viral Genomes ◽

Viral Particles ◽

Demethylase Activity ◽

Retroviral Replication ◽

Hiv 1

ABSTRACTDuring retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that HIV-1 full-length RNA packaging is regulated through an epitranscriptomic switch requiring demethylation of two conserved adenosine residues present within the 5′-UTR. As such, while m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation was required for the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and drives full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

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Electron Microscopy of a Herpesvirus Isolated from Marek's Disease in Duck and Chicken Embryo Fibroblast Cultures

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100858 ◽

1968 ◽

Vol 26 ◽

pp. 222-223 ◽

Cited By ~ 1

Author(s):

Keyvan Nazerian

Keyword(s):

Inclusion Bodies ◽

Chicken Embryo Fibroblast ◽

Marek's Disease ◽

Marek’S Disease ◽

Duck Embryo Fibroblast ◽

Multinucleated Cells ◽

Viral Particles ◽

Infected Cells ◽

And Control ◽

Do So

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

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