Rubella virus assembly requirements and evolutionary relationships with novel rubiviruses

Abstract

ABSTRACT Rubella virus (RuV) is an enveloped virus that usually causes mild disease in children, but can produce miscarriage or severe congenital birth defects. While in nature RuV only infects humans, the discovery of the related Ruhugu (RuhV) and Rustrela (RusV) viruses highlights the spillover potential of mammalian rubiviruses to humans. RuV buds into the Golgi, but its assembly and exit are not well understood. We identified a potential late domain motif 278 PPAY 281 at the C-terminus of the RuV E2 envelope protein. Such late domain motifs can promote virus budding by recruiting the cellular ESCRT machinery. An E2 Y281A mutation reduced infectious virus production by >3 logs and inhibited virus particle production. However, RuV was insensitive to inhibition by dominant-negative VPS4, and thus appeared ESCRT-independent. The E2 Y281A mutation did not significantly inhibit the production of the viral structural proteins capsid (Cp), E2, and E1, or dimerization, glycosylation, Golgi transport, and colocalization of E2 and E1. However, E2 Y281A significantly reduced glycoprotein-Cp colocalization and interaction, and inhibited Cp localization to the Golgi. Revertants of the E2 Y281A mutant contained an E2 281V substitution or the second site mutations [E2 N277I + Cp D215A]. These mutations promoted virus growth, particle production, E2/Cp colocalization and Cp-Golgi localization. Both the E2 substitutions 281V and 277I were found at the corresponding positions in the RuhV E2 protein. Taken together, our data identify a key interaction of the RuV E2 endodomain with the Cp during RuV biogenesis, and support the close evolutionary relationship between human and animal rubiviruses. IMPORTANCE Rubella virus (RuV) is an enveloped virus that only infects humans, where transplacental infection can cause miscarriage or congenital birth defects. We identified a potential late domain, 278 PPAY 281 , at the C terminus of the E2 envelope protein. However, rather than this domain recruiting the cellular ESCRT machinery as predicted, our data indicate that E2 Y281 promotes a critical interaction of the E2 endodomain with the capsid protein, leading to capsid's localization to the Golgi where virus budding occurs. Revertant analysis demonstrated that two substitutions on the E2 protein could partially rescue virus growth and Cp-Golgi localization. Both residues were found at the corresponding positions in Ruhugu virus E2, supporting the close evolutionary relationship between RuV and Ruhugu virus, a recently discovered rubivirus from bats.