Disulfide Bonds and Membrane Topology of the Vaccinia Virus A17L Envelope Protein (original) (raw)

Membrane Topology of the Vaccinia Virus A17L Envelope Protein

Virology, 1999

The formation of a lipoprotein membrane within specialized areas of the cytoplasm is the first visible step in poxvirus morphogenesis. The A17L viral protein, an essential nonglycosylated membrane component, was predicted to have four centrally located alpha-helical membrane-spanning domains. The gene was expressed as a 23-kDa protein in a cell-free transcription/translation system containing canine pancreatic microsomes. The N- and C-terminal ends of the membrane-associated protein were susceptible to proteinase digestion, whereas the central region was resistant, consistent with a model in which the first and fourth hydrophobic domains are membrane spanning. This topology was supported by the sizes of the major proteinase-resistant membrane-associated products of genes containing one or more deleted hydrophobic domains and by evidence that the C-terminus was intraluminal and glycosylated on deletion of the second, third, and fourth domains, the third and fourth domains, or just the fourth domain. Moreover, glycosylation also occurred when an N-glycosylation site was introduced into the second hydrophobic domain of the full-length A17L protein. The data indicated a predominant topology in which the N- and C-termini are cytoplasmic, the first and fourth hydrophobic domains span the microsomal membrane, and the second and third hydrophobic domains are intraluminal. This arrangement has important implications for interactions of the A17L protein with other membrane components.

Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

Journal of Virology, 2009

A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV ). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfidelinked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix

Journal of virology, 1998

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the f...

Vaccinia virus lacking A17 induces complex membrane structures composed of open membrane sheets

Archives of Virology, 2011

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640-4648, 1). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein.

Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus

Journal of virology, 1999

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thym...

Vaccinia Virus G4L Glutaredoxin Is an Essential Intermediate of a Cytoplasmic Disulfide Bond Pathway Required for Virion Assembly

Journal of Virology, 2002

Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10R SS 3 G4L SS 3 L1R SS , F9L SS ) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4-malemideylstilbene-2,2disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for trans-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli

The Journal of biological chemistry, 1990

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus pa...

The Role of a 21-kDa Viral Membrane Protein in the Assembly of Vaccinia Virus from the Intermediate Compartment

Journal of Biological Chemistry, 1996

We have recently provided morphological evidence that a key event in the assembly of vaccinia virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521-541). This tightly apposed cisternal domain incompletely surrounds the spherical immature virus that matures into the first of the two distinct infectious forms of vaccinia, the intracellular mature virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640 -4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH 2 -and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH 2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies against Rab1.

Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein

Journal of virology, 1997

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precu...