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1.
PLoS Comput Biol ; 19(8): e1011309, 2023 08.
Article in English | MEDLINE | ID: mdl-37535676

ABSTRACT

Hepatitis B virus (HBV) infection kinetics in immunodeficient mice reconstituted with humanized livers from inoculation to steady state is highly dynamic despite the absence of an adaptive immune response. To recapitulate the multiphasic viral kinetic patterns, we developed an agent-based model that includes intracellular virion production cycles reflecting the cyclic nature of each individual virus lifecycle. The model fits the data well predicting an increase in production cycles initially starting with a long production cycle of 1 virion per 20 hours that gradually reaches 1 virion per hour after approximately 3-4 days before virion production increases dramatically to reach to a steady state rate of 4 virions per hour per cell. Together, modeling suggests that it is the cyclic nature of the virus lifecycle combined with an initial slow but increasing rate of HBV production from each cell that plays a role in generating the observed multiphasic HBV kinetic patterns in humanized mice.


Subject(s)
Hepatitis B , Virus Replication , Animals , Mice , Kinetics , DNA, Viral , Hepatitis B virus/genetics , Virion/physiology
2.
J Virol ; 97(6): e0043723, 2023 06 29.
Article in English | MEDLINE | ID: mdl-37195206

ABSTRACT

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.


Subject(s)
Herpesvirus 4, Human , Secretory Vesicles , Viral Proteins , Virus Release , Virus Replication , Humans , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/physiology , Secretory Vesicles/metabolism , Secretory Vesicles/virology , Virion/physiology , Virus Replication/physiology , HEK293 Cells , Viral Proteins/metabolism , Virus Release/genetics
3.
J Cell Biol ; 222(1)2023 01 02.
Article in English | MEDLINE | ID: mdl-36250941

ABSTRACT

Virus assembly, which takes place during the late stage of viral replication, is essential for virus propagation. However, the underlying mechanisms remain poorly understood, especially for viruses with complicated structures. Here, we use correlative light and electron microscopy to examine the formation of cytoplasmic virion assembly compartments (cVACs) during infection by a γ-herpesvirus. These cVACs are membraneless organelles with liquid-like properties. Formation of cVACs during virus infection is mediated by ORF52, an abundant tegument protein. ORF52 undergoes liquid-liquid phase separation (LLPS), which is promoted by both DNA and RNA. Disrupting ORF52 phase separation blocks cVACs formation and virion production. These results demonstrate that phase separation of ORF52 is critical for cVACs formation. Our work defines herpesvirus cVACs as membraneless compartments that are generated through a process of LLPS mediated by a tegument protein and adds to the cellular processes that are facilitated by phase separation.


Subject(s)
Herpesviridae , Virion , Virus Assembly , Cytoplasm , RNA/metabolism , Virion/physiology , Viral Proteins , Organelles
4.
J Cell Sci ; 136(5)2023 03 01.
Article in English | MEDLINE | ID: mdl-36093836

ABSTRACT

Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.


Subject(s)
Kinesins , Vaccinia , Cell Extracts , Humans , Microtubules/metabolism , Vaccinia/metabolism , Vaccinia virus , Virion/physiology
5.
J Virol ; 96(22): e0107322, 2022 11 23.
Article in English | MEDLINE | ID: mdl-36300940

ABSTRACT

Tegument, which occupies the space between the nucleocapsid and the envelope, is a unique structure of a herpesvirion. Tegument proteins are major components of tegument and play critical roles in virus life cycle. Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus subfamily, is closely related to two human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We have previously shown that MHV-68 ORF33, conserved among all herpesviruses, encodes a tegument protein that is associated with intranuclear capsids and is essential for virion morphogenesis and egress. Another tegument protein ORF45, which is conserved only among gammaherpesviruses, also plays an essential role in virion morphogenesis of MHV-68. In this study, we investigated the underlying mechanism and showed that these two proteins colocalize and interact with each other during virus infection. We mapped the ORF33-interacting domain to the conserved carboxyl-terminal 23 amino acids (C23) of ORF45. Deletion of the C23 coding sequence in the context of viral genome abolished the production of infectious virions. Transmission electron microscopy results demonstrated that C23 of ORF45 are essential for virion tegumentation in the cytoplasm. We further mapped the ORF45-interacting domain to the N-terminal 17 amino acids (N17) of ORF33. Deletion of the N17 coding sequence in the context of viral genome also abolished production of infectious virions, and N17 of ORF33 are also essential for virion tegumentation in the cytoplasm. Taken together, our data strongly indicate that the interaction between ORF45 and ORF33 plays an essential role in cytoplasmic maturation of MHV-68 virions. IMPORTANCE A critical step in viral lytic replication is the assembly of progeny viral particles. Herpesviruses are important pathogens. A herpesvirus particle comprises, from inside to outside, four layers: DNA core, capsid, tegument, and envelope. The tegument layer contains dozens of virally encoded tegument proteins, which play critical roles in virus assembly. Murine gammaherpesvirus 68 (MHV-68) is a tumor-associated herpesvirus and is closely related to Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. We previously found that the absence of either tegument protein ORF33 or ORF45 inhibits the translocation of nucleocapsids to the cytoplasm and blocks virion maturation, but the underlying mechanism remained unclear. Here, we showed that ORF33 interacts with ORF45. We mapped their interaction domains and constructed viral mutants with defects in ORF33-ORF45 interaction. Transmission electron microscopy data demonstrated that the assembly of these viral mutants in the cytoplasm is blocked. Our results indicate that ORF33-ORF45 interaction is essential for gammaherpesvirus replication.


Subject(s)
Capsid Proteins , Immediate-Early Proteins , Rhadinovirus , Virus Assembly , Animals , Mice , Cytoplasm/metabolism , Herpesvirus 4, Human , Herpesvirus 8, Human , Rhadinovirus/genetics , Rhadinovirus/physiology , Virion/genetics , Virion/physiology , Virus Replication , Capsid Proteins/metabolism , Immediate-Early Proteins/metabolism
6.
Nat Commun ; 13(1): 2935, 2022 05 26.
Article in English | MEDLINE | ID: mdl-35618710

ABSTRACT

Serine Incorporator 5 (SERINC5), a cellular multipass transmembrane protein that is involved in sphingolipid and phosphatydilserine biogenesis, potently restricts a number of retroviruses, including Human Immunodeficiency Virus (HIV). SERINC5 is incorporated in the budding virions leading to the inhibition of virus infectivity. In turn, retroviruses, including HIV, encode factors that counteract the antiviral effect of SERINC5. While SERINC5 has been well studied in retroviruses, little is known about its role in other viral families. Due to the paucity of information regarding host factors targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), we evaluated the effect of SERINC proteins on SARS-CoV-2 infection. Here, we show SERINC5 inhibits SARS-CoV-2 entry by blocking virus-cell fusion, and SARS-CoV-2 ORF7a counteracts the antiviral effect of SERINC5 by blocking the incorporation of over expressed SERINC5 in budding virions.


Subject(s)
COVID-19 , HIV Infections , Antiviral Agents/pharmacology , Humans , Membrane Proteins , SARS-CoV-2 , Virion/physiology
7.
Microbiol Spectr ; 10(1): e0245221, 2022 02 23.
Article in English | MEDLINE | ID: mdl-35170992

ABSTRACT

Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm3, whereas this density has a very low viral titer in the prototypic Fermon strain. IMPORTANCE Despite the strong causal link between enterovirus D68 (EV-D68) and acute flaccid myelitis (AFM), it remains unclear how EV-D68 gains entry into the central nervous system and what receptors enable it to infect motor neurons. We show that EV-D68 particles can adhere to exosomes, placing EV-D68 among a handful of other picornaviruses which are known to interact with extracellular vesicles. Uptake and infection of permissive cells by virally contaminated exosomes would have major implications in the search for the EV-D68 receptor, as well as providing a possible route for viral entry into motor neurons. This work identifies a novel cellular entry route for EV-D68 and may facilitate the identification of genetic risk factors for development of AFM.


Subject(s)
Central Nervous System Viral Diseases/virology , Enterovirus D, Human/chemistry , Enterovirus D, Human/physiology , Enterovirus Infections/virology , Exosomes/virology , Myelitis/virology , Neuromuscular Diseases/virology , Virion/chemistry , Cell Line , Densitometry , Humans , Neurons/chemistry , Neurons/virology , Virion/physiology , Virus Internalization
8.
Viruses ; 14(2)2022 01 24.
Article in English | MEDLINE | ID: mdl-35215818

ABSTRACT

The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qß, revealing that the 3' untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA-RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.


Subject(s)
Bacteriophages/physiology , Virion/physiology , Virus Assembly , Bacteriophages/chemistry , Bacteriophages/genetics , Bacteriophages/ultrastructure , Capsid/metabolism , Capsid/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cryoelectron Microscopy , Models, Molecular , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Virion/chemistry , Virion/genetics , Virion/ultrastructure
9.
Viruses ; 14(2)2022 01 25.
Article in English | MEDLINE | ID: mdl-35215825

ABSTRACT

Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.


Subject(s)
Epithelial Cells/virology , RNA Viruses/physiology , Virus Release , Cell Polarity , Humans , Virion/physiology , Virus Assembly , Virus Replication
10.
Viruses ; 14(2)2022 01 28.
Article in English | MEDLINE | ID: mdl-35215863

ABSTRACT

Chikungunya virus (CHIKV) presents a major burden on healthcare systems worldwide, but specific treatment remains unavailable. Attachment and fusion of CHIKV to the host cell membrane is mediated by the E1/E2 protein spikes. We used an in vitro single-particle fusion assay to study the effect of the potent, neutralizing antibody CHK-152 on CHIKV binding and fusion. We find that CHK-152 shields the virions, inhibiting interaction with the target membrane and inhibiting fusion. The analysis of the ratio of bound antibodies to epitopes implied that CHIKV fusion is a highly cooperative process. Further, dissociation of the antibody at lower pH results in a finely balanced kinetic competition between inhibition and fusion, suggesting a window of opportunity for the spike proteins to act and mediate fusion, even in the presence of the antibody.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Chikungunya virus/immunology , Chikungunya virus/physiology , Virus Internalization , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Cell Line , Hydrogen-Ion Concentration , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/metabolism , Virion/physiology , Virus Attachment
11.
Viruses ; 14(2)2022 02 02.
Article in English | MEDLINE | ID: mdl-35215905

ABSTRACT

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.


Subject(s)
Erythrocytes/virology , Fish Diseases/virology , Isavirus/physiology , Orthomyxoviridae Infections/veterinary , Salmo salar/virology , Animals , Endothelial Cells/virology , Fish Diseases/blood , Isavirus/genetics , Isavirus/isolation & purification , Orthomyxoviridae Infections/blood , Orthomyxoviridae Infections/virology , Salmo salar/blood , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/genetics , Virion/isolation & purification , Virion/physiology , Virus Replication
12.
Viruses ; 14(2)2022 02 05.
Article in English | MEDLINE | ID: mdl-35215918

ABSTRACT

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.


Subject(s)
Alphavirus Infections/veterinary , Alphavirus Infections/virology , Alphavirus/physiology , Alphavirus/ultrastructure , Virus Assembly , Alphavirus/classification , Alphavirus/genetics , Animals , Cattle/virology , Cryoelectron Microscopy , Dimerization , Foxes/virology , Horses/virology , Humans , Models, Molecular , Phylogeny , Swine/virology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virion/classification , Virion/genetics , Virion/physiology , Virion/ultrastructure
13.
Viruses ; 14(2)2022 02 05.
Article in English | MEDLINE | ID: mdl-35215917

ABSTRACT

Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.


Subject(s)
Capsid/metabolism , Gene Products, gag/metabolism , Retroviridae Infections/virology , Retroviridae/physiology , Virus Assembly , Animals , Gene Products, gag/genetics , Genome, Viral , Humans , Retroviridae/genetics , Virion/genetics , Virion/physiology
14.
Viruses ; 14(2)2022 02 08.
Article in English | MEDLINE | ID: mdl-35215941

ABSTRACT

Flavivirus outbreaks require fast and reliable diagnostics that can be easily adapted to newly emerging and re-emerging flaviviruses. Due to the serological cross-reactivity among flavivirus antibodies, neutralization tests (NT) are considered the gold standard for sero-diagnostics. Here, we first established wild-type single-round infectious virus replicon particles (VRPs) by packaging a yellow fever virus (YFV) replicon expressing Gaussia luciferase (Gluc) with YFV structural proteins in trans using a double subgenomic Sindbis virus (SINV) replicon. The latter expressed the YFV envelope proteins prME via the first SINV subgenomic promoter and the capsid protein via a second subgenomic SINV promoter. VRPs were produced upon co-electroporation of replicon and packaging RNA. Introduction of single restriction enzyme sites in the packaging construct flanking the prME sequence easily allowed to exchange the prME moiety resulting in chimeric VRPs that have the surface proteins of other flaviviruses including dengue virus 1--4, Zika virus, West Nile virus, and tick-borne encephalitis virus. Besides comparing the YF-VRP based NT assay to a YF reporter virus NT assay, we analyzed the neutralization efficiencies of different human anti-flavivirus sera or a monoclonal antibody against all established VRPs. The assays were performed in a 96-well high-throughput format setting with Gluc as readout in comparison to classical plaque reduction NTs indicating that the VRP-based NT assays are suitable for high-throughput analyses of neutralizing flavivirus antibodies.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Flavivirus/immunology , High-Throughput Screening Assays/methods , Cross Reactions , Flavivirus/classification , Flavivirus/genetics , Flavivirus/physiology , Genes, Reporter , Luciferases/genetics , Luciferases/metabolism , Neutralization Tests , Replicon , Sindbis Virus/genetics , Sindbis Virus/immunology , Sindbis Virus/physiology , Virion/genetics , Virion/immunology , Virion/physiology , Yellow fever virus/genetics , Yellow fever virus/immunology , Yellow fever virus/physiology
15.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: mdl-35215979

ABSTRACT

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.


Subject(s)
Defective Viruses/physiology , Virion/physiology , Animals , Colony-Forming Units Assay , Genome, Viral , Humans , Microscopy, Electron, Transmission , Viral Plaque Assay , Virus Diseases/virology , Virus Replication
16.
Gene ; 809: 146024, 2022 Jan 30.
Article in English | MEDLINE | ID: mdl-34673207

ABSTRACT

Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6-/- mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6-/- cells significantly exceeded the virus titer in the original HEK293FT cells.


Subject(s)
Annexin A6/genetics , Host-Pathogen Interactions/genetics , Influenza A virus/physiology , Virus Replication/physiology , Annexin A6/metabolism , CRISPR-Cas Systems , Gene Knockout Techniques , HEK293 Cells , Humans , Influenza A virus/pathogenicity , Virion/physiology
17.
J Virol ; 96(4): e0183121, 2022 02 23.
Article in English | MEDLINE | ID: mdl-34878808

ABSTRACT

Most viruses undergo a maturation process from a weakly self-assembled, noninfectious particle to a stable, infectious virion. For herpesviruses, this maturation process resolves several conflicting requirements: (i) assembly must be driven by weak, reversible interactions between viral particle subunits to reduce errors and minimize the energy of self-assembly, and (ii) the viral particle must be stable enough to withstand tens of atmospheres of DNA pressure resulting from its strong confinement in the capsid. With herpes simplex virus 1 (HSV-1) as a prototype of human herpesviruses, we demonstrated that this mechanical capsid maturation is mainly facilitated through capsid binding auxiliary protein UL25, orthologs of which are present in all herpesviruses. Through genetic manipulation of UL25 mutants of HSV-1 combined with the interrogation of capsid mechanics with atomic force microscopy nano-indentation, we suggested the mechanism of stepwise binding of distinct UL25 domains correlated with capsid maturation and DNA packaging. These findings demonstrate another paradigm of viruses as elegantly programmed nano-machines where an intimate relationship between mechanical and genetic information is preserved in UL25 architecture. IMPORTANCE The minor capsid protein UL25 plays a critical role in the mechanical maturation of the HSV-1 capsid during virus assembly and is required for stable DNA packaging. We modulated the UL25 capsid interactions by genetically deleting different UL25 regions and quantifying the effect on mechanical capsid stability using an atomic force microscopy (AFM) nanoindentation approach. This approach revealed how UL25 regions reinforced the herpesvirus capsid to stably package and retain pressurized DNA. Our data suggest a mechanism of stepwise binding of two main UL25 domains timed with DNA packaging.


Subject(s)
Capsid/physiology , Herpesviridae/physiology , Virus Assembly/physiology , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA Packaging , Herpesvirus 1, Human/physiology , Humans , Microscopy, Atomic Force , Mutation , Protein Binding , Protein Domains , Virion/genetics , Virion/metabolism , Virion/physiology
18.
Med Sci (Paris) ; 38(12): 1016-1027, 2022 Dec.
Article in French | MEDLINE | ID: mdl-36692281

ABSTRACT

Viruses can provide new biological functions to plants and animals. Some viruses persisting at low levels in plants might confer resistance to stress and parasites. In animals, more numerous examples of genes originating from viruses and used by different organisms have been described. For examples these genes might contribute to protect from new infections, or to ensure communication between neurons or to enable placenta development. In parasitic wasps, a complex viral machinery has been conserved as an endogenous virus dispersed in the wasp genome, which produces virions. These virions infect the parasitized host resulting in the production of virulence factors that inhibit defense mechanisms against the parasite. Different organisms have used the same viral functions repeatedly during animal evolution.


Title: Des virus bénéfiques pour les plantes et les animaux. Abstract: Les virus peuvent apporter de nouvelles fonctions aux organismes qui les portent. Chez les plantes, des virus, présents à des niveaux d'infection faibles, confèrent des propriétés de résistance aux stress et aux parasites. Chez les animaux, de plus nombreux exemples d'appropriation de gènes viraux, qui participent en particulier à la protection contre de nouvelles infections, à la communication entre les neurones, ou à la morphogenèse du placenta, ont été décrits. Chez les guêpes parasites, une machinerie virale complexe est conservée sous la forme d'un virus endogène dispersé dans le génome, leur permettant d'infecter l'hôte parasité et de lui faire exprimer des protéines inhibant ses propres mécanismes de défense. Les processus d'appropriation des mêmes fonctions virales se sont souvent répétés au cours de l'évolution. Cette revue aborde des exemples de symbioses virales (c'est-à-dire, des cas où le virus exploite un organisme-hôte en lui étant par ailleurs bénéfique), où l'apport positif des virus est bien documenté.


Subject(s)
Polydnaviridae , Viruses , Wasps , Animals , Polydnaviridae/physiology , Viruses/genetics , Virion/physiology , Virus Physiological Phenomena , Virulence Factors
19.
Nat Commun ; 12(1): 7087, 2021 12 06.
Article in English | MEDLINE | ID: mdl-34873158

ABSTRACT

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector.


Subject(s)
Aphids/genetics , Cucumovirus/genetics , Insect Proteins/genetics , Insect Vectors/genetics , RNA, Satellite/genetics , RNA, Viral/genetics , Animals , Aphids/physiology , Aphids/virology , Cucumovirus/physiology , Gene Expression Regulation , Host-Pathogen Interactions , Insect Proteins/metabolism , Insect Vectors/physiology , Insect Vectors/virology , Plant Diseases/genetics , Plant Diseases/parasitology , Plant Diseases/virology , Plant Leaves/genetics , Plant Leaves/parasitology , Plant Leaves/virology , Plants, Genetically Modified , RNA, Satellite/physiology , RNA, Viral/physiology , Nicotiana/genetics , Nicotiana/parasitology , Nicotiana/virology , Virion/genetics , Virion/physiology , Virus Replication/genetics , Virus Replication/physiology
20.
Viruses ; 13(12)2021 11 30.
Article in English | MEDLINE | ID: mdl-34960664

ABSTRACT

Herpes simplex virus type 1, or HSV-1, is a widespread human pathogen that replicates in epithelial cells of the body surface and then establishes latent infection in peripheral neurons. When HSV-1 replicates, viral progeny must be efficiently released to spread infection to new target cells. Viral spread occurs via two major routes. In cell-cell spread, progeny virions are delivered directly to cellular junctions, where they infect adjacent cells. In cell-free release, progeny virions are released into the extracellular milieu, potentially allowing the infection of distant cells. Cell-cell spread of HSV-1 has been well studied and is known to be important for in vivo infection and pathogenesis. In contrast, HSV-1 cell-free release has received less attention, and its significance to viral biology is unclear. Here, I review the mechanisms and regulation of HSV-1 cell-free virion release. Based on knowledge accrued in other herpesviral systems, I argue that HSV-1 cell-free release is likely to be tightly regulated in vivo. Specifically, I hypothesize that this process is generally suppressed as the virus replicates within the body, but activated to high levels at sites of viral reactivation, such as the oral mucosa and skin, in order to promote efficient transmission of HSV-1 to new human hosts.


Subject(s)
Cell-Free System/virology , Herpes Simplex/transmission , Herpes Simplex/virology , Herpesvirus 1, Human/physiology , Virion/physiology , Virus Release , Animals , Cell Line , Herpesvirus 1, Human/genetics , Humans , Virion/genetics
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