Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Abstract

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This Tϭ4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3,8,24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8,19,30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18,20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1,7,14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6,13,14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9,36).In this study, we characte...

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

“…These structures contain helical tubular arrays of viral glycoproteins within the vesicles, which are studded with nucleocapsids on their cytoplasmic face (91)(92)(93). Their proximity to the PM suggests that CPV-IIs may be an assembly intermediate (92), but it is not clear whether they are necessary for efficient infection or contribute to pathogenesis. CHIKV CPV-I and CPV-II structures also have been observed in mosquito cells (91).…”

Probing the Early Temporal and Spatial Interaction of the Sindbis Virus Capsid and E2 Proteins with Reverse Genetics

“…Recent evidence has suggested that NCs and envelope proteins cotraffic from cytoplasmic domains to the plasma membrane on virus-induced membrane structures termed cytopathic vacuoles type II (CPV-II), and NC and the envelope proteins likely interact on these CPV-II structures (19,41). Furthermore, a morphologically distinct virus-induced membrane structure, termed cytopathic vacuole type I (CPV-I), likely contains the viral replicase and has been implicated as the site of initial NC assembly (11).…”

“…Soonsawad and colleagues showed that CPV-II vacuoles contain envelope proteins spikes, are decorated with NCs, and traffic from sites internal to the cell to the plasma membrane (41). Unpublished electron tomography data (T. J. Edwards and R. J. Kuhn) have demonstrated the movement of assembled NCs from CPV-I vacuoles to CPV-II vacuoles, and recent studies using microinjection of viral RNA into envelope protein-expressing cells demonstrated a preference for newly synthesized envelope proteins during envelopment of NCs (39).…”

“…These CPV-II molecules have been implicated in the traffic of envelope proteins to the plasma membrane and appear to be decorated with NCs (19,41). The precise origin of CPV-II vacuoles and the temporal and spatial aspects of the initial interaction between NC and envelope proteins on these CPV-II structures have yet to be detailed.…”

“…It is thought that the trimer of E1-E2 heterodimers, often referred to as spikes, forms a two-dimensional (2D) crystalline array at the plasma membrane and probably already arrives at the plasma membrane in a spike-like conformation (20,41,49). However, the site of the initial E1-E2 heterodimerization, as well as the subcellular compartment in which trimerization occurs, is not clear, although these processes likely occur soon after translation (34).…”