Rubella Virus: First Calcium-Requiring Viral Fusion Protein

Abstract

Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca ²⁺ during virus entry. Other tested cations did not substitute. Ca ²⁺ was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca ²⁺ was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca ²⁺-dependent viral fusion protein and has a unique membrane interaction mechanism.

Author Summary

Rubella virus (RuV) is a small enveloped RNA virus causing mild disease in children. However, infection of pregnant women can produce fetal death or congenital rubella syndrome, a constellation of severe birth defects including cataracts, hearing loss, heart disease and developmental delays. While vaccination has greatly reduced disease in the developed world, rubella remains prevalent in developing countries and other undervaccinated populations. RuV infects cells by endocytic uptake and a low pH-triggered membrane fusion reaction mediated by the viral E1 protein. The postfusion structure of E1 revealed a metal ion complexed at the membrane-interacting tip of the protein. Here we demonstrated that RuV infection and fusion are completely dependent on calcium, which could not be replaced functionally by any other metal that was tested. In the absence of calcium, RuV entry and low pH-conformational changes were unchanged, but E1's interaction with the target membrane was specifically blocked. Mutations of the calcium-binding residues in E1 caused a similar inhibition of E1 membrane interaction, fusion and infection. Thus, RuV E1 is the first known example of a calcium-dependent virus fusion protein.