Semliki forest virus budding: assay, mechanisms, and cholesterol requirement.

Virus budding is a critical step in the life cycle of all enveloped viruses. Budding may be defined as the progressive envelopment of the virus core by a cellular membrane enriched with viral membrane proteins, culminating in a membrane fission reaction to release the completed virus particle. Different viruses use different host cell membranes as budding sites, including the plasma membrane and various membranes of the exocytic pathway. Viruses also differ in their requirements for virus proteins to drive the budding reaction (14). Viruses such as the alphaviruses and hepadnaviruses have a strict requirement for both nucleocapsids and spike proteins to permit virus budding (31, 57, 59). In contrast, the budding reactions of various other viruses can be driven by the capsid or core protein, by the matrix protein, or by the membrane proteins, without obligatory involvement of the other viral protein subunits (14). Virus budding reactions are an important area of research because of their key roles in virus replication, their potential as therapeutic targets, and their relevance to cellular membrane budding reactions. However, quantitative experimental methods to specifically assay budding have been limited. In the absence of such focused systems, broader studies of infectious particle production by necessity measure a wide range of reactions during the virus life cycle, including the biosynthesis of viral components and the traffic of viral membrane proteins through the exocytic pathway. The lack of more effective model systems has made it difficult to address fundamental questions about viral budding reactions, such as requirements for cellular components and energy sources. Alphaviruses such as Semliki Forest virus (SFV) are simple, well-characterized enveloped animal viruses (see references 20 and 56 for a review). Each SFV particle contains 240 copies of four structural proteins: the capsid protein, which packages the single plus-stranded RNA genome into nucleocapsids, and three envelope proteins, the type I transmembrane polypeptides E1 and E2 (each about 50 kDa) and the peripheral E3 polypeptide (∼10 kDa). The envelope proteins assemble into 80 spikes, each consisting of a trimer, (E1/E2/E3)3. Both the spike protein layer and the viral nucleocapsid are arranged as T=4 icosohedral structures, which associate with each other via a one-to-one interaction of the E2 internal domain and the capsid protein (7, 13). The virus lipid bilayer is derived from the host cell plasma membrane during budding. The life cycle of SFV and other alphaviruses has been studied in detail (20, 56). SFV enters host cells via receptor-mediated endocytosis, and virus membrane fusion is mediated by the spike protein and triggered by the low pH present in the endosome (15, 20). In addition to its low pH requirement, fusion of alphaviruses such as SFV is also strongly dependent on the presence of cholesterol and sphingolipid in the target membrane (21, 25, 37, 38, 65). Following fusion between the viral and endosome membranes, nucleocapsids are released into the cytoplasm and viral replication is initiated. Progeny RNA molecules associate with capsid protein in the cytoplasm to form new nucleocapsids. The spike protein E1 subunit and the E2 precursor, p62, are translated and translocated into the rough endoplasmic reticulum, where they are glycosylated and form a stable but noncovalently associated heterodimer. The dimer is transported through the secretory pathway and processing of p62 to mature E2 and E3 is carried out in the late secretory pathway by furin, a cellular protease. E1, E2, and E3 are then transported to the plasma membrane, where virus budding occurs. Alphavirus budding is a clear example of a budding reaction that requires both nucleocapsids and spike proteins. This is due to a specific interaction between the capsid protein and a key tyrosine-containing motif in the cytoplasmic tail of E2 (56, 67). Structural studies indicate that this region of E2 binds to a hydrophobic pocket on the surface of the nucleocapsid (28, 52). Expression studies demonstrated that, although p62 could be transported to the cell surface in the absence of E1, stable association with nucleocapsids did not occur, suggesting that the dimeric interaction of E1 and E2 is important to maintain the correct conformation of the E2 tail for nucleocapsid binding (1). Further studies showed that lateral interactions between heterodimers are also required for efficient budding (12). In keeping with the known structure of the alphavirus particle (7, 13), it appears that the E1/E2 heterodimers assemble into trimers which associate via lateral interactions and that the resultant multivalency of E2-nucleocapsid binding promotes virus budding. In addition to these requirements for viral proteins in budding, studies of alphavirus infection in cholesterol-depleted cells have revealed a role for cholesterol in efficient virus exit. Along with their well-characterized requirement for cholesterol in the membrane fusion reaction, both SFV and Sindbis virus (SIN) show strong requirements for cholesterol in exit (32, 34). In contrast, srf-3 (sterol requirement in function), an SFV mutant selected for more efficient growth in cholesterol-depleted cells, has markedly increased levels of both fusion and exit in the absence of cholesterol compared to those of wild-type (wt) virus (34, 62). The increased cholesterol independence of srf-3 fusion and exit were demonstrated to be caused by a single point mutation in the srf-3 E1 subunit, proline 226→serine (P226S) (62). Mutagenesis studies of SIN demonstrated that the E1 226 region is also involved in the cholesterol dependence of its fusion and exit (32). However, although these studies reveal a role for cholesterol in the alphavirus exit pathway, the stage of the viral exit pathway affected and the mechanism of the cholesterol effect remain unclear. We have here established a quantitative biochemical assay for SFV budding that is generally applicable to viruses that bud from the plasma membrane. The assay was based on biotin derivatization of radiolabeled virus spike proteins at the cell surface and subsequent retrieval of biotin-tagged virions using magnetic streptavidan particles. We have used the assay to define several basic properties of SFV budding, including kinetics, temperature dependence, and the role of cytoskeletal elements and exocytic transport. Using wt virus and the srf-3 mutant, we demonstrated that the previously observed cholesterol requirement for production of wt SFV was due to a block in spike protein budding from cholesterol-depleted cells. The block in wt virus budding correlated with the rapid degradation of the wt spike proteins throughout the infectious cycle in sterol-deficient cells. (This research was conducted by Y.E.L. in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.)