Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus

Abstract

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 lacIoperator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.

Vaccinia Virus Motility

“…Among the several proteins on the IMV surface, three have been shown to bind to glycosoaminoglycans (GAGs). The A27L and H3L gene products bind to heparan sulfate (44,62,119) and the D8L protein binds to chondroitin sulfate (45). Virus binding to GAGs is only the initial interaction of the virus with the cell, and additional host and virus molecules are likely to be involved in virus penetration.…”

“…Two of these proteins, B5R and F13L, and the IMV A27L protein are needed for wrapping. A virus mutant with an A27L Ala25 to Asp substitution permitted IMV transport but not wrapping (96), and N-terminally truncated A27L does not make EEV (119). Similarly, mutant viruses lacking B5R or F13L form 5-or 10-fold less EEV owing to a defect in wrapping, and wrapping is also inhibited by the drug N 1 -isonicotinoyl-N 2 -3-methyl-4-chlorobenzoylhydrazine (IMCBH) [for review see (102)].…”

“…The third helix (residues 77-98) contains a leucine-zipper motif that is important for interacting with A17L (120). The N-terminal 20 amino acids are cleaved in the mature protein (110) to expose a lysine-and arginine-rich region (residues 21-32) that binds to heparan sulfate (44,119). The membrane fusion domain lies within residues 29-43 and the second helix is important for formation of the coiled-coil trimer (119,120).…”

Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

“…Several MV membrane proteins are known targets of neutralizing antibody: A27 [ 15 , 16 ], A28 [ 17 ], D8 [ 18 ], H3 [ 19 , 20 ] and L1 [ 21 ]. Of these proteins, A27 [ 22 - 24 ], H3 [ 19 ] and D8 [ 25 ] are involved in virus attachment and A28 [ 26 ] and L1 [ 27 ] in membrane fusion and virus entry. The MV proteins do not traffic through the secretory pathway of the cell, creating obstacles to their isolation for protein vaccines and presentation for DNA vaccines.…”

From Crescent to Mature Virion: Vaccinia Virus Assembly and Maturation

“…Endocytosis is triggered by virus-receptor interactions, followed by intracellular transportation before membrane fusion. Four viral proteins involved in VACV attachment to host cells have been identified, including A26, A27, H3, and D8, which bind with glycosaminoglycan or laminin on the cell surface [ 11 , 12 , 13 , 14 , 15 ]. Membrane fusion is believed to be mediated or catalyzed by a large macromolecular assembly of viral proteins in the MV, which has been named the entry fusion complex (EFC).…”