The Envelope of Intracellular African Swine Fever Virus Is Composed of a Single Lipid Bilayer

Abstract

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.The assembly and envelopment of the large nucleocytoplasmic DNA viruses such as African swine fever virus (ASFV) and vaccinia virus (VV) have been the subject of many studies, but the mechanisms and pathways that govern the morphogenesis of each virus still remain controversial (1,11,12,23,24). ASFV virions have a classic icosahedral structure that is similar to that of the iridoviruses, whereas VV immature virions (IV) are spherical. Despite the obvious differences in appearance between intracellular ASFV virions and VV IV, the structural organizations are similar. The center of each virus consists of the genome surrounded by a layer of protein, which in the case of ASFV is called the matrix or inner core shell. This is in turn enclosed by a lipid envelope which lies beneath the capsid (2, 3, 6, 7, 10). The immature VV matures into the brick-shaped intracellular mature virus. ASFV gains a further outer envelope as it buds through the plasma membrane on exit (21). The following experiments refer to the intracellular form of ASFV only.Considerable analysis of the envelopes of the spherical immature and brick-shaped intracellular mature forms of VV suggests that the envelope originates from membrane crescents formed in the cytoplasm near assembly sites. Early models of VV assembly proposed that the crescents and IV envelopes were single lipid bilayers formed by de novo membrane biogenesis (8). However, sequencing of the poxvirus genome did not support this initial hypothesis, as genes encoding lipid synthesis enzymes were not found (25). In addition, intracellular membranes are usually present as double-membrane cisternae, and it was unclear how the virus could stabilize single bilayers within the cellular environment. In an ...

The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event

“…This model, however, has been recently questioned, and based on data obtained using samples prepared by high-pressure freezing, it has been suggested that the inner envelope of ASFV consists of a single lipid bilayer (28). The icosahedral capsid layer, formed by protein p72, is then progressively assembled on one side of this envelope, while on the other side, the core shell domain, mainly constituted by the processing products of the polyproteins pp220 and pp62, is simultaneously constructed (6,7,20,26).…”

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid

“…Phycodnaviruses, together with mimivirus, iridoviruses, asfarviruses, ascoviruses, and poxviruses have a common evolutionary ancestor and they are among the largest and most complex viruses known. Collectively these viruses are referred to as nucleocytoplasmic large DNA viruses (NCLDV) (3)(4)(5)(6)(7)(8). With the exception of the poxviruses and ascoviruses, all of these viruses are roughly icosahedral in shape and have surfaces consisting of hexagonally closed packed arrays of capsomers organized into tri-and pentasymmetrons (SI Text).…”

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

“…The traditional view of the inner envelope as formed by two tightly opposed membranes derived from ER collapsed cisternae (7,59,60) has recently been challenged by the careful examination of the width of the internal membrane of viral particles and the single outer mitochondrial membrane, carried out using chemical fixation, cryosectioning, and high-pressure freezing (34). The results suggest that the inner envelope of ASFV is a single lipid bilayer, which raises the question of how such a structure can be generated and stabilized in the precursors of the ASFV internal envelope.…”

“…Intracellular particles are formed by an inner viral core, which contains the central nucleoid surrounded by a thick protein coat, referred to as core shell. This core is enwrapped by an inner lipid envelope (7,34) on top of which the icosahedral capsid is assembled (26,27,31). Extracellular virions possess an additional membrane acquired during the budding from the plasma membrane (11).…”