The Myristate Moiety and Amino Terminus of Vaccinia Virus L1 Constitute a Bipartite Functional Region Needed for Entry

Abstract

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for cell entry and the fusion of infected cells. L1 associates with members of the entry-fusion complex (EFC), but its specific role in entry has not been delineated. We recently demonstrated (Foo CH, et al., Virology 385:368 -382, 2009) that soluble L1 binds to cells and blocks entry, suggesting that L1 serves as the receptor-binding protein for entry. Our goal is to identify the structural domains of L1 which are essential for its functions in VACV entry. We hypothesized that the myristate and the conserved residues at the N terminus of L1 are critical for entry. To test our hypothesis, we generated mutants in the N terminus of L1 and used a complementation assay to evaluate their ability to rescue infectivity. We also assessed the myristoylation efficiency of the mutants and their ability to interact with the EFC. We found that the N terminus of L1 constitutes a region that is critical for the infectivity of VACV and for myristoylation. At the same time, the nonmyristoylated mutants were incorporated into mature virions, suggesting that the myristate is not required for the association of L1 with the viral membrane. Although some of the mutants exhibited altered structural conformations, two mutants with impaired infectivity were similar in conformation to wild-type L1. Importantly, these two mutants, with changes at A4 and A5, undergo myristoylation. Overall, our results imply dual differential roles for myristate and the amino acids at the N terminus of L1. We propose a myristoyl switch model to describe how L1 functions. P oxviruses are unique DNA viruses that replicate in the cytoplasm and employ a multifaceted form of morphogenesis (18). Vaccinia virus (VACV) is the prototype virus that has been used to study the replication of poxviruses (40, 48), and in an attenuated form the virus was deployed as a vaccine for the global eradication of smallpox (30,39). Multiple infectious forms of VACV are produced during viral replication. The majority of the infectious progeny virions are single-enveloped mature virions (MV), which form adjacent to virus replication sites and are released only by cell lysis.During the initial step of entry, the MV utilizes four envelope proteins, A27, H3, D8, and A26, to mediate attachment to a variety of cell surface molecules, such as heparan sulfate proteoglycan (HSPG) (16, 44), chondroitin sulfate proteoglycan (34), and the extracellular matrix protein laminin (15). After adsorption, the virion can use pH-dependent or pH-independent pathways to enter cells (6,14,47,60,83). The specific viral proteins responsible for receptor binding, virus internalization, the triggering of viruscell fusion, and membrane fusion itself are poorly defined. Components of a conserved, multiprotein entry-fusion complex (EFC) are proposed to perform certain nonredundant functions that are critical for entry (8, 11, 38, 52, 53, 61, 64-66, 76-78, 85). Eleven proteins have thus far been identified to form the massive complex ...

Protein Primary Structure of the Vaccinia Virion at Increased Resolution

“…We first examined fatty acylation as one of the known posttranslational modifications of vaccinia virus (see the introduction). One of the best known acylations in vaccinia virus is the N-terminal myristoylation of L1 protein (7,8,36). In the current study, a myristoylation mark was found at the N-terminal Gly of the initiator methionine-demethionylated Nterminal tryptic peptide of vaccinia virus protein L1R (sucrose preparation) and nowhere else in this proteome.…”

The 2.1 Å structure of protein F9 and its comparison to L1, two components of the conserved poxvirus entry-fusion complex

“…Greater distances between α-carbons up to 3.84 Å RMSD were only found in loops. Phe108, located at the end of the cavity in L1 and required for structural integrity 33 , is conserved in all L1 orthologs except SGPV097.…”

“…A hydrophobic cavity capable of accepting and shielding a conjugated myristic acid is located near the N-terminus 32 . Myristoylation of L1 is required for infectivity but not for assembly of the EFC and incorporation into viral particles 33 . The amino acid motif for adding a myristate moiety is conserved in all poxvirus orthologs of L1, except in Salmon gill poxvirus (SGPV) 21 .…”

Membrane fusion during poxvirus entry

“…There is a large hydrophobic cavity that could accommodate the N-terminal myristate moiety [47] although this has not been demonstrated. Mutations within the cavity inhibit infectivity without affecting myristoylation [72]. A “myristate switch” model in which the acyl chain is released from the cavity during entry has been proposed.…”