Vaccinia Virus G9 Protein Is an Essential Component of the Poxvirus Entry-Fusion Complex

Abstract

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.The mechanisms by which enveloped DNA viruses enter cells are poorly understood compared to those for many enveloped RNA viruses (17). Entry of the latter is typically mediated by one or two viral glycoproteins and involves virus attachment to the cell, activation of a fusion protein, and ultimately, merging of the viral and cellular membranes to allow entry of the genome and associated proteins. In contrast, three to four glycoproteins are required for entry of herpesviruses (35) and even more are needed for entry of vaccinia virus (VACV), the prototype poxvirus (26). Studies of VACV entry have been complicated by the existence of two infectious forms: the mature virion (MV), which contains a nucleoprotein core surrounded by a membrane containing more than 20 nonglycosylated proteins, and the extracellular virion (EV), which is essentially an MV surrounded by an additional membrane containing five proteins that are glycosylated and one that is not (9,26,34). The MV, which is extremely stable and can be liberated by cell lysis, is thought to mediate transmission between host animals, whereas the EV mediates cell-to-cell spread. There is evidence that the MV and EV bind differently to cells (39), consistent with their different outer membrane proteins. Binding of the MV to some cells appears to be due at least in part to three membrane proteins that can bind heparan or chondroitin sulfate (7,19,20,23,40), although individ...

Redundancy and Plasticity of Neutralizing Antibody Responses Are Cornerstone Attributes of the Human Immune Response to the Smallpox Vaccine

“…Nondisulfide-bonded forms of the L1 and F9 proteins were also made, but these exhibited no serological reactivity, indicating that correct folding was important for their immunogenicity (data not shown). Two additional MV surface proteins have since been identified, and these were not included in the synthesized microarrays (9,40,42,43,48,54). Use of protein microarrays to measure antibody levels has found useful applications in diverse fields during the past 5 years (12,49).…”

Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein

“…We found that F9-deficient MVs could bind to cells but that cytoplasmic cores were not detected by antibody labeling. A similar phenotype was found for other protein components of the putative entry/fusion complex (22,23,30,32,36,37). F9 shares many characteristics with components of the entry/fusion complex including (i) conservation among all sequenced poxviruses, (ii) postreplicative expression, (iii) association with the MV membrane, (iv) formation of intramolecular disulfide bonds by the poxvirus-encoded redox pathway, and (v) absence of an essential role in morphogenesis.…”

“…Initial experiments indicated that the protein was associated with MVs purified by sucrose density gradient centrifugation. Compared to L1, F9 was poorly soluble upon extraction with NP-40 or NP-40 plus DTT (data not shown) as found for some components of the entry/fusion complex (22,23). Evidence for the location of F9 in the MV membrane was obtained by surface biotinylation of purified MVs.…”

“…Those proteins associated with the MV membrane can be placed into at least four functional groups involved in membrane crescent formation, e.g., A14 and A17 (25,27,38,41); virion maturation, e.g., A9 and L1 (24,43); virus attachment to glycosaminoglycans, e.g., H3 and D8 (14,18); and virus entry, e.g., A16, A21, A28, G9, H2, and L5 (22,23,30,32,36). When expression of any one of the aforementioned entry proteins is repressed, typical-looking MVs and EVs form but they are defective in entry and induction of syncytia after brief low-pH treatment.…”

Electron tomography of viruses

“…Historically, these studies have spanned both interactions of viruses with antibodies as well as studies by electron [27][28][29] and light microscopy [30] of various stages in the viral life cycle. Recent progress in both areas provides encouraging signs that the use of 3D electron microscopic approaches could also be invaluable in the physiological context.…”