Hand-foot-and-mouth disease (HFMD) is a serious public health threat across the Asia-Pacific region, as evidenced by the more than 1.7 million cases that were reported by the Chinese Ministry of Health in China during 2010. The dominant causative agent is EV71, a non-enveloped single-stranded RNA virus (genus Enterovirus,
) closely related to Coxsackievirus A16, the other etiological agent of HFMD1
. Whilst A16 infections are not usually serious, acute EV71 infections may cause severe neurological disease2,3
, leading to 905 deaths in China in 2010.
The icosahedral capsid of EV71 comprises 60 copies of four protein subunits, VP1-VP4. During assembly the P1 polyprotein is cleaved to yield VP0 (36KD), VP1 (32KD) and VP3 (27KD). The final cleavage of VP0 into VP2 (28KD) and VP4 (8KD), autocatalysed by viral RNA, forms the mature viral capsid4,5
, in which VP1-VP3 follow a pseudo T=3 arrangement and span the thickness of the capsid6
, whilst VP4 is located on the inside. Picornaviruses may also produce empty particles, which resemble the mature virus in structure and antigenicity, sediment at ~73S and comprise 60 copies of VP0, VP1 and VP34,7,8
. In-line with this, EV71 cultured in Vero cells for vaccine development produces two distinct types of particles9
: highly infectious mature virions, composed of VP1-4 and RNA, and empty particles containing VP0, VP1 and VP3. Although 73S particles are no longer thought to be direct precursors of mature virions, they can function as reservoirs of capsid components8,10,11
, presumably following their dissociation into functional subunits. 73S particles are rather unstable, readily converting from D-type native antigenicity to a state with altered (C-type in the poliovirus nomenclature12,13
) antigenic properties.
Structural studies have outlined the processes leading to cell infection by enteroviruses14
. Cellular receptors attach to the virus, often binding in a canyon-like depression surrounding the 5-fold axis15-17
. This triggers conformational changes in the virus, leading to the formation of an expanded intermediate with altered antigenic properties, which sediments at ~135S (cf
~160S for the mature virus)18-20
. This conformational change (which can also be induced by environmental insults such as heating or low pH), leads to the externalisation of VP4 and the VP1 N termini21,22
, followed by extrusion of the viral genome into the cytoplasm of the target cell, to leave an ~80S empty particle21
. From low-resolution data, the consensus view was that the viral RNA probably exited via a 5-fold axis channel14
, although recent studies suggest it exits near a 2-fold axis23
. There remains a dearth of high-resolution structural detail to illuminate the molecular mechanisms underpinning this dynamic cell-entry process. We have determined structures of both inactivated and infectious EV71 virions, and of expanded natural empty particles closely resembling expanded enterovirus uncoating intermediates previously visualized by cryo-electron microscopy (cryo-EM)24,25
. Our high-resolution analyses suggest a detailed molecular mechanism for the early stages of enterovirus uncoating.