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Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

ABSTRACT: Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- and caveolae-mediated endocytosis, and the internalization of TGEVs was almost completed within ~2 minutes after TGEVs attached to the cell membrane. Furthermore, the interactions of TGEVs with actin and dynamin 2 in real time during the TGEV internalization were visualized. To our knowledge, this is the first report that single-virus tracking technique is used to visualize the entire dynamic process of the TGEV internalization: before the TGEV internalization, with the assistance of actin, clathrin, and caveolin 1 would gather around the virus to form the vesicle containing the TGEV, and after ~60 seconds, dynamin 2 would be recruited to promote membrane fission. These results demonstrate that TGEVs enter ST cells via clathrin- and caveolae-mediated endocytic, actin-dependent, and dynamin 2-dependent pathways.

SUBMITTER: Wang J

PROVIDER: S-EPMC7163995 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

Wang Jian J Li Yangyang Y Wang Shouyu S Liu Fei F

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20200204 3

Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- ...[more]

PMID: 32017270

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Project description:UnlabelledChikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes.ImportanceSince its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, conflicting results with regard to the cell entry pathway used by CHIKV have been published. Here we applied a novel technology to visualize the entry behavior of single CHIKV particles in living cells. Our results show that CHIKV cell entry is extremely rapid and occurs via clathrin-mediated endocytosis. Membrane fusion from within acidic early endosomes is observed. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taking these findings together, this study provides detailed insight into the cell entry process of CHIKV.

Project description:We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600-10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected approximately 36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident approximately 18 h postinfection. Leader-containing GFP transcripts containing the ORF 3B and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines.

Project description:UnlabelledPorcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are economically important swine enteropathogenic coronaviruses. These two viruses belong to two distinct species of the Alphacoronavirus genus within Coronaviridae and induce similar clinical signs and pathological lesions in newborn piglets, but they are presumed to be antigenically distinct. In the present study, two-way antigenic cross-reactivity examinations between the prototype PEDV CV777 strain, three distinct U.S. PEDV strains (the original highly virulent PC22A, S indel Iowa106, and S 197del PC177), and two representative U.S. TGEV strains (Miller and Purdue) were conducted by cell culture immunofluorescent (CCIF) and viral neutralization (VN) assays. None of the pig TGEV antisera neutralized PEDV and vice versa. One-way cross-reactions were observed by CCIF between TGEV Miller hyperimmune pig antisera and all PEDV strains. Enzyme-linked immunosorbent assays, immunoblotting using monoclonal antibodies and Escherichia coli-expressed recombinant PEDV and TGEV nucleocapsid (N) proteins, and sequence analysis suggested at least one epitope on the N-terminal region of PEDV/TGEV N protein that contributed to this cross-reactivity. Biologically, PEDV strain CV777 induced greater cell fusion in Vero cells than did U.S. PEDV strains. Consistent with the reported genetic differences, the results of CCIF and VN assays also revealed higher antigenic variation between PEDV CV777 and U.S. strains.ImportanceEvidence of antigenic cross-reactivity between porcine enteric coronaviruses, PEDV and TGEV, in CCIF assays supports the idea that these two species are evolutionarily related, but they are distinct species defined by VN assays. Identification of PEDV- or TGEV-specific antigenic regions allows the development of more specific immunoassays for each virus. Antigenic and biologic variations between the prototype and current PEDV strains could explain, at least partially, the recurrence of PEDV epidemics. Information on the conserved antigenicity among PEDV strains is important for the development of PEDV vaccines to protect swine from current highly virulent PEDV infections.

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Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

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Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

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