Orsay δ Protein Is Required for Nonlytic Viral Egress

Abstract

Nonenveloped gastrointestinal viruses, such as human rotavirus, can exit infected cells from the apical surface without cell lysis. The mechanism of such nonlytic exit is poorly understood. The nonenveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans Dye staining results suggested that Orsay virus exits from the intestine of infected worms in a nonlytic manner. Therefore, the Orsay virus-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay virus genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein, δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had a normal CP-δ fusion but could not produce free δ due to the lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and colocalized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay virus infection rearranged ACT-5 apical localization. Reduction of the ACT-5 level via RNA interference (RNAi) significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay virus exit. Together, these data support a model in which the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate the polarized, nonlytic egress of Orsay virus.IMPORTANCE An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain nonenveloped viruses can exit cultured cells in nonlytic ways; however, such nonlytic exit has not been demonstrated in vivo In addition, it is not clear how such nonlytic exit is achieved mechanistically in vivo Orsay virus is a nonenveloped RNA virus that infects the intestine cells of the nematode C. elegans It is currently the only virus known to naturally infect C. elegans Using this in vivo model, we show that the δ protein encoded by Orsay virus facilitates the nonlytic exit of the virus, possibly by interacting with host actin on the apical side of worm intestine cells.

Keywords: Caenorhabditis elegans; Orsay; viral egress.

FIG 1

Orsay virus has nonlytic exit. Shown is propidium iodide staining of uninfected (A), Cry5B-treated (B), and Orsay virus-infected (C) animals. From left to right, the images show PI staining, merged PI staining, and Nomarski images, with merged PI staining and autofluorescence in the blue channel. *, intracellular regions that were not stained by PI. Bar, 10 μm.

FIG 2

The ATG mutant virus has exit defects. (A) Schematic drawing of the strategy for the generation of recombinant virus. (B) qRT-PCR results showing viral loads in infected worms and in culture media. Data were obtained from three biological replicates, each with three technical replicates. Bars and error bars show means and standard errors (SE). **, P < 0.01 by Student's t test. (C) Viral infection kinetics based on F26F2.1p::GFP expression in infected worms (n ≥ 529 worms for each time point). Data were compiled from three independent trials with six plates for each virus genotype in each trial. (D) Viral titers of the worm lysate over storage time. The titer was normalized so that the fresh lysate (day 1) had a relative titer of 1. (E) Schematic drawing of the single-worm infection assay. (F) Single-worm infection assay results comparing the ATG mutant virus and the wild type. Data were obtained from four independent trials, each with six plates for each time point. The graph displays means and SE.

FIG 3

Free δ is localized near the apical membrane in intestine cells. (A to E) Nomarski and florescence images showing the subcellular localizations of GFP-tagged δ (A), CP-δ (B), CP (C), N-terminal aa 1 to 66 of δ (D), and C-terminal aa 67 to 346 of δ (E). Arrowheads indicate apical and basolateral membrane localizations. * indicates nuclear localization. Arrows indicate aggregates. (F) Colocalization of δ and ACT-5. The schematic drawing shows ACT-5 localization in an intestine cell. Bar, 10 μm.

FIG 4

Orsay virus infection rearranges the ACT-5 actin structure. (A) YFP::ACT-5 localization at the apical membrane is weakened 50 h after Orsay virus infection. Bar, 100 μm. (B) Percentages of animals with weak YFP::ACT-5 in uninfected worms, worms infected with the wild-type virus, and worms infected with the ATG mutant virus. ***, P < 0.001 by Fisher's exact test (n ≥ 125 animals for each group). (C) Western blotting. Tubulin was used as a loading control. The graph displays means and standard errors of data from two biological replicates, each with five technical replicates. N.S., not significant. (D to I) Orsay virus infection causes ACT-5 to have branches and gaps. (D and E) Twenty-four hours after Orsay virus infection, YFP::ACT-5 branches appear in anterior cells of infected (E) but not uninfected (D) worms. (F and G) Fifty hours after infection, YFP::ACT-5 branches appear in cells close to the midbody of infected (G) but not uninfected (F) worms. (H and I) Also 50 h after infection, branches (H) and gaps (I) can be observed in mCherry::ACT-5. Bar, 10 μm. (J) Percentages of animals with abnormal mCherry::ACT-5 in uninfected and infected worms. **, P < 0.01 by Fisher's exact test (n ≥ 69 animals for each group). For quantification purposes, if a worm showed multiple phenotypes, it was classified in the category of its most severe phenotype, following the phenotypic severity order of gap > branch > weak (from severe to weak).

FIG 5

Free δ has biological functions. (A) δ genetically interacts with act-5. Single-worm infection assay results show the effects of the δ ATG mutation in the virus and act-5(RNAi) on hosts. Data are from four independent trials, each with six plates for each genotype. The graph displays means and SE. N.S., not significant. *, P < 0.05 by Student's t test. (B) Nomarski images showing distal tip cell (DTC) migration defects in some worms upon the heat shock-induced overexpression of δ::GFP. Bar, 10 μm. Schematic line drawings under the images show the DTC migration path. (C) Single-worm infection assay results showing that the effects of expressing δ::GFP in host cells rescued the viral exit defects of the δ ATG mutant virus. Data are from five independent trials, with 6 or 12 plates for each genotype in each trial. The graph displays means and SE. *, P < 0.05 by Student's t test.

FIG 6

Proposed model of δ functions. δ may function in both viral entry and exit. In the CP-δ fusion, δ may mediate viral entry, possibly by interacting with cell surface receptors. The free δ may interact with ACT-5 to promote polarized viral exit. Proteins are color-coded: orange represents the pentameric δ fiber, green highlights the Orsay virus capsid, and red indicates ACT-5.