Entry-fusion Complex Research Articles

Identification of a Potential Entry-Fusion Complex Based on Sequence Homology of African Swine Fever and Vaccinia Virus.

African swine fever virus (ASFV) belongs to the family of Asfarviridae, part of the group of nucleocytoplasmic large DNA viruses (NCLDV). Little is known about the internalization of ASFV in the host cell and the fusion membrane events that take place at early stages of the infection. Poxviruses, also members of the NCLDV and represented by vaccinia virus (VACV), are large, enveloped, double-stranded DNA viruses. Poxviruses were considered unique in having an elaborate entry-fusion complex (EFC) composed of 11 highly conserved proteins integrated into the membrane of mature virions. Recent advances in methodological techniques have again revealed several connections between VACV EFC proteins. In this study, we explored the possibility of an analogous ASFV EFC by identifying ten candidate proteins exhibiting structural similarities with VACV EFC proteins. This could reveal key functions of these ASFV proteins, drawing attention to shared features between the two virus families, suggesting the potential existence of an ASFV entry-fusion complex.

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex.

Vaccinia virus infection requires virus-cell membrane fusion to complete entry during endocytosis; however, it contains a large viral fusion protein complex of 11 viral proteins that share no structure or sequence homology to all the known viral fusion proteins, including type I, II, and III fusion proteins. It is thus very challenging to investigate how the vaccinia fusion complex works to trigger membrane fusion with host cells. In this study, we crystallized the ectodomain of vaccinia H2 protein, one component of the viral fusion complex. Furthermore, we performed a series of mutational, biochemical, and molecular analyses and identified two surface loops containing 170LGYSG174 and 125RRGTGDAW132 as the A28-binding region. We also showed that residues in the N-terminal helical region (amino acids 51-90) are also important for H2 function.

Structural and functional analysis of vaccinia viral fusion complex component protein A28 through NMR and molecular dynamic simulations.

Host cell entry of vaccinia virus (a poxvirus) proceeds through multiple steps that involve many viral proteins to mediate cell infection. Upon binding to cells, vaccinia virus membrane fuses with host membranes via a viral entry fusion protein complex comprising 11 proteins: A16, A21, A28, F9, G3, G9, H2, J5, L1, L5 and O3. Despite vaccinia virus having two infectious forms, mature and enveloped, that have different membrane layers, both forms require an identical viral entry fusion complex for membrane fusion. Components of the poxvirus entry fusion complex that have been structurally assessed to date share no known homology with all other type I, II and III viral fusion proteins, and the large number of fusion protein components renders it a unique system to investigate poxvirus-mediated membrane fusion. Here, we determined the NMR structure of a truncated version of vaccinia A28 protein. We also expressed a soluble H2 protein and showed that A28 interacts with H2 protein at a 1:1 ratio in vitro. Furthermore, we performed extensive in vitro alanine mutagenesis to identify A28 protein residues that are critical for H2 binding, entry fusion complex formation, and virus-mediated membrane fusion. Finally, we used molecular dynamic simulations to model full-length A28-H2 subcomplex in membranes. In summary, we characterized vaccinia virus A28 protein and determined residues important in its interaction with H2 protein and membrane components. We also provide a structural model of the A28-H2 protein interaction to illustrate how it forms a 1:1 subcomplex on a modeled membrane.

Tailoring multivalent nanomedicines for monkeypox

We have been through the largest monkeypox (Mpox) outbreak that has ever occurred globally. At present, the application of nanotechnology-based approaches to tackle Mpox has not been established. There are no US FDA-approved treatments for Mpox and it is plausible that, in forthcoming studies, researchers may investigate the use of multivalent nanomaterials to target the virus. In contrast to monovalent ligands, multi- and polyvalent ligands have strong affinity for the target molecules, thereby significantly facilitating the inhibition of viral attachment to host cells. Multivalent interactions are crucial in signal processing, adhesion, and recognition within biological systems. Based on the understanding of the development of multivalent nanomaterials (such as phage capsid nanoparticles, engineered bacteriophage T4 and self-assembled DNA nanobait) for SARS-CoV-2, influenza, and other viruses, this brief perspective gives insights into the development of next-generation multivalent nanomedicines targeting the entry-fusion complex of Mpox as a promising strategy for tackling emerging mutated strains.

Widespread Distribution and Evolution of Poxviral Entry-Fusion Complex Proteins in Giant Viruses.

Poxviruses are known to encode a set of proteins that form an entry-fusion complex (EFC) to mediate virus entry. However, the diversity, evolution, and origin of these EFC proteins remain poorly understood. Here, we identify the EFC protein homologs in poxviruses and other giant viruses of the phylum Nucleocytoviricota. The 11 EFC genes are present in almost all poxviruses, with the two smallest, G3 and O3, being absent in Entomopoxvirinae and basal lineages of Chordopoxvirinae. Five of the EFC genes are further grouped into two families, A16/G9/J5 and F9/L1, which are widely distributed across other major lineages of Nucleocytoviricota, including metagenome-assembled genomes, but are generally absent in viruses infecting algae or nonamoebozoan heterotrophic protists. The A16/G9/J5 and F9/L1 families cooccur, mostly as single copies, in 93% of the non-Poxviridae giant viruses that have at least one of them. Distribution and phylogenetic patterns suggest that both families originated in the ancestor of Nucleocytoviricota. In addition to the Poxviridae genes, homologs from each of the other Nucleocytoviricota families are largely clustered together, suggesting their ancient presence and vertical inheritance. Despite deep sequence divergences, we observed noticeable conservation of cysteine residues and predicted structures between EFC proteins of Poxviridae and other families. Overall, our study reveals widespread distribution of these EFC protein homologs beyond poxviruses, implies the existence of a conserved membrane fusion mechanism, and sheds light on host range and ancient evolution of Nucleocytoviricota. IMPORTANCE Fusion between virus and host membranes is critical for viruses to release genetic materials and to initiate infection. Whereas most viruses use a single protein for membrane fusion, poxviruses employ a multiprotein entry-fusion complex (EFC). We report that two major families of the EFC proteins are widely distributed within the virus phylum Nucleocytoviricota, which includes poxviruses and other double-stranded (dsDNA) giant viruses that infect animals, amoebozoans, algae, and various microbial eukaryotes. Each of these two protein families is structurally conserved, traces its origin to the root of Nucleocytoviricota, was passed down to the major subclades of Nucleocytoviricota, and is retained in most giant viruses known to infect animals and amoebozoans. The EFC proteins therefore represent a potential mechanism for virus entry in diverse giant viruses. We hypothesize that they may have facilitated the infection of an animal/amoebozoan-like host by the last Nucleocytoviricota common ancestor.

Articles of Significant Interest In This Issue

Poxviruses are exceptional in having a multicomponent entry-fusion complex (EFC) that is comprised of eleven conserved proteins embedded in the membrane of mature virions. Schin et al. (e00852-21) adapted the tripartite split green fluorescent protein complementation system to determine individual EFC protein contacts by proximity analysis within living cells. Previous EFC protein interactions were confirmed and new ones discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins, suggesting that multiple copies stabilize the complex within the viral membrane.

Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP β-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

Loss of the vaccinia virus 35-amino acid hydrophobic O3 protein is partially compensated by mutations in the transmembrane domains of other entry proteins.

Eleven highly conserved proteins comprise the poxvirus entry-fusion complex (EFC). We focused on vaccinia virus (VACV) O3, a 35-amino acid, largely hydrophobic component of unknown specific function. Experimental evolution was carried out by blindly passaging a virus that was severely impaired in entry due to deletion of the gene encoding O3. Large plaque variants that arose spontaneously were discerned by round four and their numbers increased thereafter. Genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. There were frame-shift mutations in consecutive Ts in open reading frames F9L and D8L and a nonsynonymous base substitution in L5R. F9 and L5 are EFC proteins and D8 is involved in VACV cell attachment. The F9L mutation occurred by round four in each of three independant passages, whereas the L5R and D8L mutations were detected only after nearly all of the genomes already had the F9L mutation. Viruses with deletions of O3L and single or double F9L, L5R and D8L mutations were constructed by homologous recombination. In a single round of infection, viruses with adaptive mutations including F9L alone or in combination exhibited statistically significant higher virus titers than the parental O3L deletion mutant or the L5R or D8L mutants, consistent with the order of selection during the passages. Further analyses indicated that the adaptive F9L mutants also had higher infectivities, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3.IMPORTANCE Entry into cells is an essential first step in virus replication and an important target of vaccine- elicited immunity. For enveloped viruses, this step involves the fusion of viral and host membranes to form a pore allowing entry of the genome and associated proteins. Poxviruses are unique in that this function is mediated by an entry-fusion complex (EFC) of eleven transmembrane proteins rather than by one or a few. The large number of proteins has hindered investigation of their individual roles. We focused on O3, a predominantly hydrophobic 35 amino acid component of the vaccinia virus EFC, and found that spontaneous mutations in the transmembrane domains of certain other entry proteins can partially compensate for the absence of O3. The mutants exhibited increased infectivity, entry and assembly or stability of the EFC.

NMR assignments of vaccinia virus protein A28: an entry-fusion complex component.

Vaccinia virus (VACV) belonging to the poxvirus family enters the host cell via two different entry pathways; either endocytosis or virus/host cell membrane fusion. With respect to the virus/host cell membrane fusion, there are eleven viral membrane proteins forming a complicated entry-fusion complex (EFC), including A28, A21, A16, F9, G9, G3, H2, J5, L5, L1 and O3, to conduct the fusion function. These EFC components are highly conserved in all poxviruses and each of them is essential and necessary for the fusion activity. So far, with the exceptions of L1 and F9 whose crystal structures were reported, the structural information about other EFC components remains largely unclear. We aim to conduct a structural and functional investigation of VACV virus-entry membrane protein A28. In this work, we expressed and purified a truncated form of A28 (14kDa; residues 38-146, abbreviated as tA28 hereinafter), with deletion of its transmembrane domain (residues 1-22) and a hydrophobic segment (residues 23-37). And the assignments of its backbone and side chain 1H, 13C and 15N chemical shifts of tA28 are reported. The secondary structure propensity from TALOS+ indicates that tA28 does contain three α-helices, six β-strands and connecting loops. Aside from this, we demonstrated that tA28 does interact with fusion suppressor viral protein A26 (residues 351-500) by the 1H-15N HSQC spectrum. We interpret that A28 binding to A26 deactivates EFC fusion activity. The current study provides a valuable framework towards further structural analyses of this protein and for better understanding virus/host cell membrane fusion mechanism in association with virus entry.

Mutations Near the N Terminus of Vaccinia Virus G9 Protein Overcome Restrictions on Cell Entry and Syncytium Formation Imposed by the A56/K2 Fusion Regulatory Complex.

The entry/fusion complex (EFC) consists of 11 conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here, we describe serially passaging VACV in nonpermissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection, indicating the occurrence and enrichment of adaptive mutations. Sequencing of genomes of passaged and cloned viruses revealed mutations near the N terminus of the G9 open reading frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His-to-Arg mutation at amino acid 44 or a duplication of amino acids 26 to 39. An adaptive Tyr-to-Cys substitution at amino acid 42 was discovered using error-prone PCR to generate additional mutations. Myristoylation of G9 was unaffected by the near-N-terminal mutations. The roles of the G9 mutations in enhancing plaque size were validated by homologous recombination. The mutants exhibited enhanced entry and spread in A56/K2 cells and induced syncytia at neutral pH in HeLa cells despite the expression of A56/K2. The data suggest that the mutations perturb the interaction of G9 with A56/K2, although some association was still detected in detergent-treated infected cell lysates.IMPORTANCE The entry of enveloped viruses is achieved by the fusion of viral and cellular membranes, a critical step in infection that determines host range and provides targets for vaccines and therapeutics. Poxviruses encode an exceptionally large number of proteins comprising the entry/fusion complex (EFC), which enables infection of diverse cells. Vaccinia virus (VACV), the prototype member of the poxvirus family, also encodes the fusion regulatory proteins A56 and K2, which are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are nonpermissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2.

Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex.

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

The 2.1 \xc5 structure of protein F9 and its comparison to L1, two components of the conserved poxvirus entry-fusion complex

The poxvirus F9 protein is a component of the vaccinia virus entry fusion complex (EFC) which consists of 11 proteins. The EFC forms a unique apparatus among viral fusion proteins and complexes. We solved the atomic structure of the F9 ectodomain at 2.10 Å. A structural comparison to the ectodomain of the EFC protein L1 indicated a similar fold and organization, in which a bundle of five α-helices is packed against two pairs of β-strands. However, instead of the L1 myristoylation site and hydrophobic cavity, F9 possesses a protruding loop between α-helices α3 and α4 starting at Gly90. Gly90 is conserved in all poxviruses except Salmon gill poxvirus (SGPV) and Diachasmimorpha longicaudata entomopoxvirus. Phylogenetic sequence analysis of all Poxviridae F9 and L1 orthologs revealed the SGPV genome to contain the most distantly related F9 and L1 sequences compared to the vaccinia proteins studied here. The structural differences between F9 and L1 suggest functional adaptations during evolution from a common precursor that underlie the present requirement for each protein.

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes.IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry.

A Novel Mode of Poxvirus Superinfection Exclusion That Prevents Fusion of the Lipid Bilayers of Viral and Cellular Membranes

Superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. The vaccinia virus A56 and K2 proteins in the cell membrane can prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. Here, we described another form of exclusion that is established earlier in infection and does not require the A56 or K2 protein. Cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia virus particles. A related orthopoxvirus, but neither a flavivirus nor a rhabdovirus, was also excluded, indicating selectivity. Although superinfecting vaccinia virus bound to cells, infection was inhibited at the membrane fusion step, thereby preventing core entry into the cytoplasm and early gene expression. In contrast, A56/K2 protein-mediated exclusion occurred subsequent to membrane fusion. Induction of resistance to superinfection depended on viral RNA and protein synthesis by the primary virus but did not require DNA replication. Although superinfection resistance correlated with virus-induced changes in the cytoskeleton, studies with mutant vaccinia viruses indicated that the cytoskeletal changes were not necessary for resistance to superinfection. Interferon-inducible transmembrane proteins, which can inhibit membrane fusion in other viral systems, did not prevent vaccinia virus membrane fusion, suggesting that these interferon-inducible proteins are not involved in superinfection exclusion. While the mechanism remains to be determined, the early establishment of superinfection exclusion may provide a "winner-take-all" reward to the first poxvirus particles that successfully initiate infection and prevent the entry and genome reproduction of defective or less fit particles. The replication of a virus usually follows a defined sequence of events: attachment, entry into the cytoplasm or nucleus, gene expression, genome replication, assembly of infectious particles, and spread to other cells. Although multiple virus particles may enter a cell at the same time, mechanisms exist to prevent infection by subsequent viruses. The latter phenomenon, known as superinfection exclusion, can occur by a variety of mechanisms that are not well understood. We showed that superinfection by vaccinia virus was prevented at the membrane fusion step, which closely followed virion attachment. Thus, neither gene expression nor genome replication of the superinfecting virus occurred. Expression of early proteins by the primary virus was necessary and sufficient to induce the superinfection-resistant state. Superinfection exclusion may be beneficial to vaccinia virus by selecting particles that can infect cells rapidly, excluding defective particles and synchronizing the replication cycle.

Preparation of truncated orf virus entry fusion complex proteins by chemical synthesis

Members of the Chordopoxvirinae subfamily possess an unusual 11 protein entry-fusion complex (EFC) that is highly conserved and present in all species. The mode of action of this EFC is unknown, and the interactions of the constituent proteins are uncharacterised. Here, we present the chemical synthesis of membrane domain truncated linear constructs of two EFC proteins in orf virus, ORFV036 and 049. By using Boc solid phase peptide synthesis and native chemical ligation methods, these truncated proteins have been readily prepared in milligram quantities. These robust synthetic protocols allow ready access to these polypeptides to facilitate biological studies.

Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry.

Analysis of viral membranes formed in cells infected by a vaccinia virus L2-deletion mutant suggests their origin from the endoplasmic reticulum.

Assembly of the poxvirus immature virion (IV) membrane is a poorly understood event that occurs within the cytoplasm. At least eight viral proteins participate in formation of the viral membrane. Of these, A14, A17, and D13 are structural components whereas A6, A11, F10, H7, and L2 participate in membrane biogenesis. L2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (ER) throughout the cell and at the edges of crescent-shaped IV precursors. Previous studies with an inducible L2 mutant revealed abortive formation of the crescent membrane. However, possible low-level L2 synthesis under nonpermissive conditions led to ambiguity in interpretation. Here, we constructed a cell line that expresses L2, which allowed the creation of an L2-deletion mutant. In noncomplementing cells, replication was aborted prior to formation of mature virions and two types of aberrant structures were recognized. One consisted of short crescents, at the surface of dense masses of viroplasm, which were labeled with antibodies to the A11, A14, A17, and D13 proteins. The other structure consisted of "empty" IV-like membranes, also labeled with antibodies to the viral proteins, which appeared to be derived from adjacent calnexin-containing ER. A subset of 25 proteins examined, exemplified by components of the entry-fusion complex, were greatly diminished in amount. The primary role of L2 may be to recruit ER and modulate its transformation to viral membranes in juxtaposition with the viroplasm, simultaneously preventing the degradation of viral proteins dependent on viral membranes for stability.

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

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.