scholarly journals Microfilaments and Microtubules Alternately Coordinate the Multistep Endosomal Trafficking of Classical Swine Fever Virus

2021 ◽  
Vol 95 (10) ◽  
Author(s):  
Yan Cheng ◽  
Jin-xiu Lou ◽  
Chun-chun Liu ◽  
Ya-yun Liu ◽  
Xiong-nan Chen ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Classical Swine Fever ◽  
Early Endosome ◽  
Fever Virus ◽  
Late Endosome ◽  
Host Cells ◽  
Target Cells ◽  
Dynamic Changes

ABSTRACT The cytoskeleton, as a ubiquitous structure in the cells, plays an important role in the processes of virus entry, replication, and survival. However, the action mechanism of the cytoskeleton in the invasion of pestivirus into host cells remains unclear. In this study, we systematically dissected the key roles of the main cytoskeleton components, namely microfilaments and microtubules, in the endocytosis of the porcine pestivirus classical swine fever virus (CSFV). We observed the dynamic changes of actin filaments in CSFV entry. Confocal microscopy showed that CSFV invasion induced the dissolution and aggregation of stress fibers, resulting in the formation of lamellipodia and filopodia. Chemical inhibitors and RNA interference were used to find that the dynamic changes of actin were caused by an EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42-cofilin signaling pathway, which regulates the microfilaments to help CSFV entry. Furthermore, colocalization of the microfilaments with clathrin and Rab5 (early endosome), as well as that of microtubules with Rab7 (late endosome) and Lamp1 (lysosome) revealed that microfilaments were activated and rearranged to help CSFV trafficking to the early endosome after endocytosis. Subsequently, recruitment of microtubules by CSFV also assisted membrane fusion of the virions from the late endosome to the lysosome with the help of a molecular motor, dynein. Unexpectedly, vimentin, which is an intermediate filament, had no effect on CSFV entry. Taken together, our findings comprehensively revealed the molecular mechanisms of cytoskeletal components that regulated CSFV endocytosis and facilitated further understanding of pestivirus entry, which would be conducive to exploration of antiviral molecules to control classical swine fever. IMPORTANCE Endocytosis, an essential biological process mediating cellular internalization events, is often exploited by pathogens for their entry into target cells. Previously, we reported different mechanisms of CSFV endocytosis into the porcine epithelial cells (PK-15) and macrophages (3D4/21); however, the details of microfilaments/microtubules mediated virus migration within the host cells remained to be elucidated. In this study, we found that CSFV infection induced rearrangement of actin filaments regulated by cofilin through an EGFR-PI3K/MAPK-RhoA/Rac1/Cdc42 pathway. Furthermore, we found that CSFV particles were trafficked along actin filaments in early and late endosomes, and through microtubules in lysosomes after entry. Here, we provide for the first time a comprehensive description of the cytoskeleton that facilitates the entry and the intracellular transport of a highly pathogenic swine virus. Results from this study will greatly contribute to the understanding of virus-induced early and complex changes in host cells that are important in CSFV pathogenesis.

scholarly journals The ESCRT-I Subunit Tsg101 Plays Novel Dual Roles in Entry and Replication of Classical Swine Fever Virus

2020 ◽  
pp. JVI.01928-20
Author(s):  
Chun-chun Liu ◽  
Ya-yun Liu ◽  
Yan Cheng ◽  
Yun-Na Zhang ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Classical Swine Fever Virus ◽  
Molecular Mechanisms ◽  
Animal Health ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Regulatory Role ◽  
High Morbidity ◽  
Replication Process

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the highly contagious diseases of swine with high morbidity and mortality, that negatively affects the pig industry worldwide, in particular China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. Endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological, metabolic processes including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT regulates the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in Clathrin-mediated endocytosis of CSFV and also involved in CSFV trafficking. Tsg101 assisted the virus in entering the host cell through the late endosome (Rab7 and Rab9), and finally to reach the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and dsRNA or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not Golgi in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genus Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both Clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for Pestivirus inhibitors.

scholarly journals Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Camille Melissa Johnston ◽  
Ulrik Fahnøe ◽  
Louise Lohse ◽  
Jens Bukh ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Population Change ◽  
Virulent Strain ◽  
Severe Disease ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Virus Population ◽  
Highly Virulent

ABSTRACT Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

40. Interactions between Classical swine fever virus and host cells

2002 ◽  
Vol 72 ◽  
pp. 14
Author(s):  
E. Bensaude ◽  
D. Sweetman ◽  
D.J. Paton ◽  
T.W. Drew ◽  
T. Wileman ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells

scholarly journals Different evolutionary patterns of classical swine fever virus envelope proteins

2016 ◽  
Vol 62 (3) ◽  
pp. 210-219 ◽  
Author(s):  
Yan Li ◽  
Zexiao Yang ◽  
Mingwang Zhang
Keyword(s):  
Classical Swine Fever Virus ◽  
Functional Divergence ◽  
Classical Swine Fever ◽  
Acid Sites ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Fever Virus ◽  
Host Cells ◽  
Virus Envelope ◽  
Evolutionary Patterns

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, which is a highly contagious disease of the domestic pig as well as wild boar. The proteins Erns, E1, and E2 are components of the viral envelope membrane. They are also implicated in virus attachment and entry, replication, and (or) anti-immune response. Here, we studied the genetic variations of these envelope proteins in the evolution of CSFV. The results reveal that the envelope proteins underwent different evolutionary fates. In Ernsand E1, but not E2, a number of amino acid sites experienced functional divergence. Furthermore, the diversification in Ernsand E1 was generally episodic because the divergence-related changes of E1 only occurred with the separation of 2 major groups of CSFV and that of Ernstook place with the division of 1 major group. The major divergence-related sites of Ernsare located on one of the substrate-binding regions of the RNase domain and C-terminal extension. These functional domains have been reported to block activation of the innate immune system and attachment and entry into host cells, respectively. Our results may shed some light on the divergent roles of the envelope proteins.

scholarly journals The Npro product of classical swine fever virus interacts with IκBα, the NF-κB inhibitor

2008 ◽  
Vol 89 (8) ◽  
pp. 1881-1889 ◽  
Author(s):  
Virginie Doceul ◽  
Bryan Charleston ◽  
Helen Crooke ◽  
Elizabeth Reid ◽  
Penny P. Powell ◽  
...  
Keyword(s):  
Immune Response ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Nuclear Accumulation ◽  
Yeast Two Hybrid ◽  
Necrosis Factor Alpha ◽  
Induced Apoptosis ◽  
Two Hybrid

Classical swine fever virus (CSFV) belongs to the genus Pestivirus and is the causative agent of classical swine fever, a haemorrhagic disease of pigs. The virus replicates in host cells without activating interferon (IFN) production and has been reported to be an antagonist of double-stranded RNA-induced apoptosis. The N-terminal protease (Npro) of CSFV is responsible for this evasion of the host innate immune response. In order to identify cellular proteins that interact with the Npro product of CSFV, a yeast two-hybrid screen of a human library was carried out, which identified IκBα, the inhibitor of NF-κB, a transcription factor involved in the control of apoptosis, the immune response and IFN production. The Npro–IκBα interaction was confirmed using yeast two-hybrid analysis and additional co-precipitation assays. It was also shown that Npro localizes to both the cytoplasmic and nuclear compartments in stably transfected cells and in CSFV-infected cells. Following stimulation by tumour necrosis factor alpha, PK-15 cell lines expressing Npro exhibited transient nuclear accumulation of pIκBα, but no effect of CSFV infection on IκBα localization or NF-κB p65 activation was observed.

scholarly journals Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

2004 ◽  
Vol 78 (16) ◽  
pp. 8812-8823 ◽  
Author(s):  
H. G. P. van Gennip ◽  
A. C. Vlot ◽  
M. M. Hulst ◽  
A. J. de Smit ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Reverse Genetics ◽  
Animal Experiments ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Target Cells ◽  
Adaptive Mutations

ABSTRACT Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476S variant into a His residue did not affect virulence. However, the 710His in the C1.1.1/CoBrB virus, together with adaptive mutations 276R, 476R, and 477I in Erns, resulted in reduced virulence in pigs. These results indicated that mutations in Erns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 Erns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

scholarly journals Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus

2002 ◽  
Vol 83 (3) ◽  
pp. 551-560 ◽  
Author(s):  
Elisenda Armengol ◽  
Karl-Heinz Wiesmüller ◽  
Daniel Wienhold ◽  
Mathias Büttner ◽  
Eberhard Pfaff ◽  
...  
Keyword(s):  
T Cell ◽  
Mhc Class Ii ◽  
Classical Swine Fever Virus ◽  
Cell Epitope ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Target Cells ◽  
T Cell Epitopes ◽  
Structural Protein

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446–1460 of the CSFV non-structural protein NS2–3 could induce interferon-γ secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

scholarly journals Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

2007 ◽  
Vol 81 (7) ◽  
pp. 3087-3096 ◽  
Author(s):  
Oliver Bauhofer ◽  
Artur Summerfield ◽  
Yoshihiro Sakoda ◽  
Jon-Duri Tratschin ◽  
Martin A. Hofmann ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Interferon Regulatory Factor ◽  
Fever Virus ◽  
Target Cells ◽  
Regulatory Factor ◽  
Cmv Promoter ◽  
Interferon Regulatory Factor 3

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

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