Virion glycosylation governs integrity and infectivity of infectious pancreatic necrosis virus

Abstract

The possible importance of the O-linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O-glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re-isolated after rate-zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re-isolation of viruses, a 3-5 (10)log-unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re-evaluation of reading values from UV-spectra of purified IPNV yielded a specific infectivity of 3 × 10(11) TCID(50)-units mg(-1) of protein and a ratio of 40 virions per TCID(50)-unit in the CHSE-214 cell system.

Figure 1

Rate‐zonal sedimentation in glycerol gradients of glycosidase‐treated virions of infectious pancreatic necrosis virus (IPNV). Purified virions metabolically radiolabelled with ³²P‐orthophosphate and ³H‐amino acids were stored in TNE. Virus was transferred into 50 mm phosphate buffer, pH 5.5, and 12.7 μg of IPNV was processed for each series as described in Materials and methods. (a) Mock‐treated sample in 50 mm phosphate buffer, pH 5.5. (b) Virions pretreated with neuraminidase followed by O‐glycosidase (NA/O). (c) Negative control of virions treated with PNGase F (PNGF) where no effect should be observed. (d) Starting material stored in TNE buffer. Sedimentation was from right to left.

Figure 2

Infectivity titrations of infectious pancreatic necrosis virus in the TNE‐ and stabilizing buffer‐buffer systems following the cycles of freeze‐thawing (F‐T). Viruses were treated and analysed as described in Materials and methods.

Figure 3

Rate‐zonal sedimentation in glycerol gradients of glycosidase‐treated infectious pancreatic necrosis virus (IPNV). Purified IPNV, metabolically radiolabelled with ³²P‐orthophosphate and ³H‐amino acids was stored in the stabilizing buffer (SB) system. Prior to enzymatic treatment virions (12.5 μg per sample) were transferred into 50 mm phosphate buffer, pH 5.5. (a) Mock‐treated series, (b) virus treated with neuraminidase followed by O‐glycosidase (NA/O), (c) virus treated with PNGase F (PNGF) as a negative control and (d) starting material maintained in SB. Sedimentation was from right to left.

Figure 4

Rate‐zonal centrifugation in glycerol gradients of periodate‐treated virions of infectious pancreatic necrosis virus. Purified and metabolically ³H‐amino acid‐labelled virions (1.8 μg of total protein in each series) were treated with sodium periodate as described in Materials and methods. The control series contained mock‐treated virions in 50 mm phosphate buffer of pH 5.5, and in the NaIO₄ series, the agent was administered at concentrations indicated in the panels. Sedimentation was from right to left. NB: The lower recoveries of radioactivity from the dense CsCl cushions are because of quenching by the CsCl because samples were counted directly (5 min) in liquid scintillation cocktail.

Figure 5

Compilation of data from five separate rate‐zonal centrifugation analyses of periodate‐treated virions of infectious pancreatic necrosis virus. One series of ³H‐mannose‐labelled (Exp. #1) and four series of ³H‐amino acid‐labelled (Exp. #2–5) viruses were treated with sodium periodate as indicated and analysed as described in Materials and methods. The relative distribution of radioactivity recovered as distinct peaks or regions in each gradient is shown as columns in the figure. As an example of sedimentation, the radioactivity profile of experiment #5 is shown in Fig. 4. The following periodate concentrations were not tested: in exp. #1; 10, 25, 50 mm NaIO4, in exp. #2; 5 mm NaIO4, in exp. #3; 25 and 50 mm NaIO4 and in exp. #4; 50 mm.