Akabane virus utilizes alternative endocytic pathways to entry into mammalian cell lines - PubMed (original) (raw)
Akabane virus utilizes alternative endocytic pathways to entry into mammalian cell lines
Norasuthi Bangphoomi et al. J Vet Med Sci. 2014 Nov.
Abstract
The entry mechanisms of Akabane virus (AKAV), Bunyaviridae family, have not yet been determined. In this study, chemical inhibitors were used to analyze endocytic mechanisms during AKAV infection of mammalian cell lines. The analyses using drug treatments followed by quantitative measurement of viral RNA and N protein revealed that AKAV enters non-bovine-derived cell lines (Vero, HmLu-1 and BHK cells) in a manner indicative of clathrin endocytosis. By contrast, AKAV infection in bovine-derived cell lines (LB9.K and MDBK cells) is independent of this pathway. Further analyses indicated that AKAV entry into bovine cell lines involves a non-clathrin, non-caveolae endocytic pathway that is dependent on dynamin. We conclude that although both cell types require a low pH for AKAV penetration, AKAV utilizes alternative entry pathways into mammalian cell lines.
Figures
Fig. 1.
Requirement of clathrin-endocytosis in AKAV entry. **(**A) LB9.K cells were treated with CPZ or without drug (control) for 1 hr following incubation with transferring (Tf) conjugated with Texas red. (B) Various cell lines were pretreated with CPZ or without drug (control) and infected with AKAV. Intracellular AKAV RNAs at 6 hr post-infection (p. i.) were measured by real-time qRT-PCR. (C) LB9.K cells were pretreated with various concentrations of CPZ or sucrose (0.45M), followed by infection with AKAV or BVDV. Then, AKAV RNAs were quantified. (D) Cells were pretreated with CPZ or without drug (control), followed by AKAV infection. At 1.5 hr p.i., cells were fixed and immunostained for AKAV N protein (green). (E) Localization of AKAV N (green) and clathrin heavy chain (CHC) marker (red) in various cell lines was immunostained. qRT-PCR values to measure AKAV RNAs in drug-treated cells are expressed as percentages relative to drug-untreated control cells. Values represent means and standard deviation (SD) of three independent experiments. Statistical significance is indicated by * (P<0.05), ** (P<0.01) or *** (P<0.005) (Student’s_t_-test).
Fig. 2.
Requirement of caveolae/lipid raft endocytosis in AKAV entry. (A) Cells were pretreated with MβCD or Nys, or without drugs (control) for 1 hr, followed by incubation with cholera toxin B subunit (CTB) conjugated with FITC. (B) Vero and LB9.K cells were pretreated with MβCD or Nys for 1 hr and infected with AKAV. Cells were incubated in the presence of drugs for 6 hr. qRT-PCR values to measure AKAV RNAs are expressed as percentages relative to untreated virus-infected control cells. Values represent means and SD of three independent experiments. Statistical significance is indicated by *** (P<0.005) (Student’s _t_-test). (C) Cells were treated with MβCD for 1 hr following AKAV infection in the presence of drug. At 1.5 hr p.i., cells were fixed and immunostained for AKAV N (green). (D) Localization of AKAV N protein (green) and caveolae marker (CAV-1) (red) in various cell was immunostained following AKAV infection at 1.5 hr p. i..
Fig. 3.
Requirement of dynamin in AKAV entry as a cellular factor in endocytic pathways. (A) LB9.K cells were pretreated with Dyn or without drug (control) for 1 hr followed by incubation with transferrin (Tf) conjugated with Texas red (red) or CTB conjugated with FITC (green). (B) LB9.K and Vero cells were pretreated with Dyn. After 1 hr of treatment, cells were infected with AKAV in the presence of drug for 6 hr. Real-time qRT-PCR values to measure intracellular AKAV RNAs are expressed as percentages relative to untreated virus-infected control cells. Values represent the mean and SD of three independent experiments. Statistical significance is indicated by *** (P<0.005) (Student’s _t_-test).
Fig. 4.
Low pH requirement of AKAV penetration. LB9.K and Vero cells were pretreated with NH4Cl or BafA1. After 1 hr of treatment, cells were infected with AKAV and were incubated in the presence of drugs for 6 hr. Real-time qRT-PCR values to measure intracellular AKAV RNAs are expressed as percentages relative to untreated virus-infected cells. Values represent the mean and SD of three independent experiments. Statistical significance is indicated by *** (P<0.005) (Student’s _t_-test).
Fig. 5.
Requirement of clathrin-endocytosis in entry of AKAV strains. LB9.K and Vero cells were pretreated with CPZ for 1 hr, followed by infection with either AKAV OBE-1 (genogroup II) or Iriki (genogroup I) strains in the presence or absence (control) of drug for 6 hr. Real-time qRT-PCR values to measure intracellular AKAV RNAs are expressed as percentages relative to untreated virus-infected cells. Values represent the mean and SD of three independent experiments. Statistical significance is indicated by *** (P<0.005) (Student’s_t_-test).
References
- Akashi H., Kaku Y., Kong X. G., Pang H.1997. Sequence determination and phylogenetic analysis of the Akabane bunyavirus S RNA genome segment. J. Gen. Virol. 78: 2847–2851. - PubMed
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