Structure-Function Analysis of Two Interacting Vaccinia Proteins That Are Critical for Viral Morphogenesis: L2 and A30.5.

An enduring mystery in poxvirology is the mechanism by which virion morphogenesis is accomplished. A30.5 and L2 are two small regulatory proteins that are essential for this process. Previous studies have shown that vaccinia A30.5 and L2 localize to the ER and interact during infection, but how they facilitate morphogenesis is unknown. To interrogate the relationship between A30.5 and L2, we generated inducible complementing cell lines (CV1-HA-L2; CV1-3xFLAG-A30.5) and deletion viruses (vΔL2; vΔA30.5). Loss of either protein resulted in a block in morphogenesis and a significant (>100-fold) decrease in infectious viral yield. Structure-function analysis of L2 and A30.5, using transient complementation assays, identified key functional regions in both proteins. A clustered charge-to-alanine L2 mutant (L2-RRD) failed to rescue a vΔL2 infection and exhibits a significantly retarded apparent molecular weight in vivo (but not in vitro), suggestive of an aberrant posttranslational modification. Furthermore, an A30.5 mutant with a disrupted putative N-terminal α-helix failed to rescue a vΔA30.5 infection. Using our complementing cell lines, we determined that the stability of A30.5 is dependent on L2 and that wild-type L2 and A30.5 coimmunoprecipitate in the absence of other viral proteins. Further examination of this interaction, using wild-type and mutant forms of L2 or A30.5, revealed that the inability of mutant alleles to rescue the respective deletion viruses is tightly correlated with a failure of L2 to stabilize and interact with A30.5. L2 appears to function as a chaperone-like protein for A30.5, ensuring that they work together as a complex during viral membrane biogenesis. IMPORTANCE Vaccinia virus is a large, enveloped DNA virus that was successfully used as the vaccine against smallpox. Vaccinia continues to be an invaluable biomedical research tool in basic research and in gene therapy vector and vaccine development. Although this virus has been studied extensively, the complex process of virion assembly, termed morphogenesis, still puzzles the field. Our work aims to better understand how two small viral proteins that are essential for viral assembly, L2 and A30.5, function during early morphogenesis. We show that A30.5 requires L2 for stability and that these proteins interact in the absence of other viral proteins. We identify regions in each protein required for their function and show that mutations in these regions disrupt the interaction between L2 and A30.5 and fail to restore virus viability.

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes.

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

The Vaccinia virion: Filling the gap between atomic and ultrastructure.

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an 'attachment protein' sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.

Entry and Disassembly of Large DNA Viruses: Electron Microscopy Leads the Way.

Nucleocytoplasmic large DNA viruses are a steadily growing group of viruses that infect a wide range of hosts and are characterized by large particle dimensions and genome sizes. Understanding how they enter into the host cell and deliver their genome in the cytoplasm is therefore particularly intriguing. Here, we review the current knowledge on the entry of two of the best-characterized nucleocytoplasmic large DNA viruses: the poxvirus Vaccinia virus (VACV) and the giant virus Mimivirus. While previous studies on VACV had proposed both direct fusion at the plasma membrane and endocytosis as entry routes, more recent biochemical and morphological data argue for macropinocytosis as well. Notably, direct imaging by electron microscopy (EM) also supported the existence of parallel ways of entry for VACV. Instead, all the giant viruses studied so far only enter cells by phagocytosis as observed by EM, and we discuss the mechanisms for opening of the particle, fusion of the viral and phagosomal membranes and genome delivery via a unique portal, specific for each giant virus. VACV core uncoating, in contrast, remains a morphologically ill-defined process. We argue that correlated light and electron microscopy methods are required to study VACV entry and uncoating in a direct and systematic manner. Such EM studies should also address whether entry of single particles and viral aggregates is different and thus provide an explanation for the different modes of entry described in the literature.

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication.

Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication.IMPORTANCE Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.

Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants.

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

HIV transmitted/founder vaccines elicit autologous tier 2 neutralizing antibodies for the CD4 binding site.

Here we report the construction, antigenicity and initial immunogenicity testing of DNA and modified vaccinia Ankara (MVA) vaccines expressing virus-like particles (VLPs) displaying sequential clade C Envelopes (Envs) that co-evolved with the elicitation of broadly neutralizing antibodies (bnAbs) to the CD4 binding site (CD4bs) in HIV-infected individual CH0505. The VLP-displayed Envs showed reactivity for conformational epitopes displayed on the receptor-binding form of Env. Two inoculations of the DNA-T/F vaccine, followed by 3 inoculations of the MVA-T/F vaccine and a final inoculation of the MVA-T/F plus a gp120-T/F protein vaccine elicited nAb to the T/F virus in 2 of 4 rhesus macaques (ID50 of ~175 and ~30). Neutralizing Ab plateaued at 100% neutralization and mapped to the CD4bs like the bnAbs elicited in CH0505. The nAb did not have breadth for other tier 2 viruses. Immunizations with T/F followed by directed-lineage vaccines, both with and without co-delivery of directed-lineage gp120 boosts, failed to elicit tier 2 neutralizing Ab for the CD4bs. Thus, pulsed exposures to DNA and MVA-expressed VLPs plus gp120 protein of a T/F Env can induce autologous tier 2 nAbs to the CD4bs.

Vaccinia virus A11 is required for membrane rupture and viral membrane assembly.

Although most enveloped viruses acquire their membrane from the host by budding or by a wrapping process, collective data argue that nucleocytoplasmic large DNA viruses (NCLDVs) may be an exception. The prototype member of NCLDVs, vaccinia virus (VACV) may induce rupture of endoplasmic-reticulum-derived membranes to build an open-membrane sphere that closes after DNA uptake. This unconventional membrane assembly pathway is also used by at least 3 other members of the NCLDVs. In this study, we identify the VACV gene product of A11, as required for membrane rupture, hence for VACV membrane assembly and virion formation. By electron tomography, in the absence of A11, the site of assembly formed by the viral scaffold protein D13 is surrounded by endoplasmic reticulum cisternae that are closed. We use scanning transmission electron microscopy-electron tomography to analyse large volumes of cells and demonstrate that in the absence of A11, no open membranes are detected. Given the pivotal role of D13 in initiating VACV membrane assembly, we also analyse viral membranes in the absence of D13 synthesis and show that this protein is not required for rupture. Finally, consistent with a role in rupture, we show that during wild-type infection, A11 localises predominantly to the small ruptured membranes, the precursors of VACV membrane assembly. These data provide strong evidence in favour of the unusual membrane biogenesis of VACV and are an important step towards understanding its molecular mechanism.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

The sleeping beauty kissed awake: new methods in electron microscopy to study cellular membranes.

Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM. The past 15-20 years are hallmarked by a tremendous interest in EM, driven by important technological advances. Cryo-EM, in particular, is now capable of revealing structures of proteins at a near-atomic resolution owing to improved sample preparation methods, microscopes and cameras. In this review, we focus on the challenges associated with the imaging of membranes by EM and give examples from the field of host-pathogen interactions, in particular of virus-infected cells. Despite the advantages of imaging membranes under native conditions in cryo-EM, conventional EM will remain an important complementary method, in particular if large volumes need to be imaged.

The scanning electron microscope in microbiology and diagnosis of infectious disease.

Despite being an excellent tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. Here we describe rapid methods that allow SEM imaging of fully hydrated, unfixed microbes without using conventional sample preparation methods. We demonstrate improved ultrastructural preservation, with greatly reduced dehydration and shrinkage, for specimens including bacteria and viruses such as Ebola virus using infiltration with ionic liquid on conducting filter substrates for SEM.

An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.

In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function.

Incomplete but infectious vaccinia virions are produced in the absence of oncolysis in feline SCCF1 cells.

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.

Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization.

The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.

Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid.

UNLABELLED: Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. IMPORTANCE: The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

Initial characterization of vaccinia virus B4 suggests a role in virus spread.

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus.

Crystal structure of the vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminal domain.

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A201₋50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A201₋50 clearly behaves as a heterodimer. The crystal structure of D4/A201₋50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A201₋50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A201₋50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A201₋50 interaction. Finally, we propose a model of D4/A201₋50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface.

Novel insights on the progression of intermediate viral forms in the morphogenesis of vaccinia virus.

Morphogenesis of vaccinia virus (VACV) is a complex structural process in which the capture of all cytoplasmic stages is difficult due to the rapid transition between the different viral forms. Taking advantage of two VACV mutants (M65 and M101) with defined genetic alterations, we described by transmission electron microscopy (TEM) of ultrathin sections novel potential transition viral forms (Ts) with reorganization of the immature virus (IV) membrane and construction of the internal core, and illustrated the envelopment steps from the mature virus (MV) to the wrapped virus (WV) stages. Our observations allowed us to propose a sequence of structural events for VACV assembly that provides key clues about VACV morphogenesis.

Cdc42 and the Rho GEF intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation.

Vaccinia virus enhances its cell-to-cell spread by inducing Arp2/3-dependent actin polymerisation. This process is initiated by Src- and Abl-mediated phosphorylation of the viral transmembrane protein A36, leading to recruitment of a signalling network consisting of Grb2, Nck, WIP and N-WASP. Nck is a potent activator of N-WASP-Arp2/3-dependent actin polymerisation. However, recent observations demonstrate that an interaction between Nck and N-WASP is not required for vaccinia actin tail formation. We found that Cdc42 cooperates with Nck to promote actin tail formation by stabilising N-WASP beneath the virus. Cdc42 activation is mediated by the Rho guanine-nucleotide-exchange factor (GEF) intersectin-1 (ITSN1), which is recruited to the virus prior to its actin-based motility. Moreover, Cdc42, ITSN1 and N-WASP function collaboratively in a feed-forward loop to promote vaccinia-induced actin polymerisation. Outside the context of infection, we demonstrate that ITSN1 also functions together with Cdc42, Nck and N-WASP during phagocytosis mediated by the Fc gamma receptor. Our observations suggest that ITSN1 is an important general regulator of Cdc42-, Nck- and N-WASP-dependent actin polymerisation.