Rescue of infectivity by in vitro transcapsidation of rotavirus single-shelled particles

Abstract

We investigated the possibility of rescuing the infectivity of noninfectious single-shelled rotavirus particles by in vitro transcapsidation. The soluble outer capsid proteins VP4 and VP7 were prepared by EDTA treatment of double-shelled (ds) particles of SA11-4F (G serotype 3), followed by removal of single-shelled (ss) particles by three sequential rounds of centrifugation. Ss-particles of B223 (G serotype 10) were prepared by two cycles of EDTA treatment of ds-particles followed by iospycnic CsCl gradient purification. A trace of infectivity (< 1000 PFU/ml) was always detected in the preparations of ss-particles, while no detectable infectivity (< 5 PFU/ml) was present in the preparations of outer capsid proteins. By mixing soluble outer capsid proteins VP4 and VP7 purified from SA11-4F and ss-particles of B223 at acidic pH (5.4), ds-like, transcapsidated particles were obtained. The transcapsidated particles were indistinguishable from genuine ds-particles by negative stain electron microscopy. However, the particles had a density intermediate between that of ds- and ss-particles. Protease-resistance studies revealed that VP7 was assembled onto transcapsidated particles in a resistant (native) form, but VP4 associated with the particles was completely protease sensitive. Viral infectivity was rescued by in vitro transcapsidation as indicated by a 500- to 1000-fold increase over background. The increased infectivity was neutralized by antiserum against SA11 (outer capsid donor), but not by antiserum against B223 (ss-particle donor). The transcapsidated particles formed small plaques like the B223 parent, and all the infectious progeny viruses contained the B223 genome. These results strongly indicate that the observed increase of infectivity was the result of in vitro transcapsidation.