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Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV., (© 2024. The Author(s).)

Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses., (© 2024. The Author(s).)

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.

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m 7 G-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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 tRNA Gln . 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 American Society for Microbiology.)

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., Competing Interests: The authors declare no conflict of interest.

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.

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., (© 2017 John Wiley & Sons Ltd.)

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.

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., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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.

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., (Published by Elsevier Inc.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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.

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an 'open-eight' conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis., (© 2013 John Wiley & Sons Ltd.)

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle., (© 2012 Blackwell Publishing Ltd.)

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.

Three-dimensional (3D) electron microscopy (EM) has become a major player in structural cell biology as it enables the analysis of subcellular architecture at an unprecedented level of detail. Interpretation of the resulting 3D volumes strongly depends on segmentation, which consists in decomposing the volume into their structural components. The computational approaches proposed so far have not turned out to be of general applicability. Thus, manual segmentation still remains a prevalent method. Here, a new computational framework for segmentation of 3D EM datasets is introduced. It relies on detection and characterization of ridges (i.e. local maxima). The detected ridges are modelled as asymmetric Gaussian functions whose parameters constitute ridge descriptors. This local information is then used to cluster the ridges, which leads to the ultimate segmentation. In this work we focus on membranes and locally planar structures in general. The performance of the framework is illustrated with its application to a number of complex 3D datasets and a quantitative analysis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The study of host-pathogen interactions over past decades has benefited from advances in microscopy and fluorescent imaging techniques. A particularly powerful model in this field is vaccinia virus (VACV), which due to its amenability to genetic manipulation has been a productive model in advancing the understanding of the transport of subcellular cargoes. Conventional light microscopy imposes an upper limit of resolution of ~250nm, hence knowledge of events occurring at the sub-viral resolution is based predominantly on studies utilising electron microscopy. The development of super-resolution light microscopy presents the opportunity to bridge the gap between these two technologies. This report describes the analysis of VACV replication using fluorescent recombinant viruses, achieving sub-viral resolution with three-dimensional structured illumination microscopy. This is the first report of successfully resolving poxvirus particle morphologies at the scale of single virus particles using light microscopy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns several directions: virus attenuation, insertion of immunomodulatory protein genes, and anti-tumor protein genes. Thymidine kinase and viral growth factor genes make the greatest contribution to the virus attenuation as their inactivation results in the virus inability to replicate in non-dividing cells, thereby contributing to increased selectivity with respect to tumor cells. Among the immunomodulatory proteins, interleukins 2, 12, and granulocyte-macrophage colony-stimulating factor proved to be most promising for oncolytic virotherapy. An attempt to use p53 protein gene expressed by vaccinia virus for addressed apoptosis of tumor cells was reported. The use of the double and triple viral recombinants carrying genes of multidirectional action seems to be most promising. Encouraging results were obtained using vaccinia virus in the oncotherapy with prodrugs and angiogenesis inhibitors. At present, two poxviral strains are undergoing Phase III clinical trials as anti-tumor preparations in the USA.

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors.

Electron tomography allows three-dimensional visualization of cellular landscapes in molecular detail. Segmentation is a paramount stage for the interpretation of the reconstructed tomograms. Although several computational approaches have been proposed, none has prevailed as a generic method and thus segmentation through manual annotation is still a common choice. In this work we introduce a segmentation method targeted at membranes, which define the natural limits of compartments within biological specimens. Our method is based on local differential structure and on a Gaussian-like membrane model. First, it isolates information through scale-space and finds potential membrane-like points at a local scale. Then, the structural information is integrated at a global scale to yield the definite segmentation. We show and validate the performance of the algorithm on a number of tomograms under different experimental conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-beta-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N' terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85alpha(-/-)beta(-/-)) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85alpha(-/-)beta(-/-) cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses.

Characterization of field isolates of viruses associated with pox-like outbreaks involving both cows (cattle) and buffaloes was carried out. PCR and electron microcopy of representative virus isolates from these animals, initially identified them as orthopoxviruses (OPXVs). Sequence and phylogenetic analyses of A-type inclusion and haemagglutinin (HA) genes of these isolates revealed a closer relationship with other OPXVs. Sequencing of the HA gene of these isolates revealed sequence identity of 96.2-99.8 and 94.6-98.7% at the nucleotide and deduced amino acid level, respectively, with VACVs, particularly with VACV-MVA, a vaccine strain. Further, C18L (ankyrin repeat protein)-gene-based BPXV-specific PCR confirmed them as BPXV. It is apparent from this study that pox-like outbreaks in cows and buffaloes in India are caused, in most cases, by BPXV. Considering the zoonotic implications of buffalopox, such outbreaks involving both buffaloes and cows in a mixed flock may pose a significant public health threat. Transmissibility of BPXV between different species including cows, buffaloes and human beings implies the potential reemergence of the virus in the subcontinent, similar to vaccinia-like outbreaks witnessed recently in other countries.

Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250x270x360nm(3). The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.

Since 1999, human infection caused by Orthopoxvirus has been observed in at least eight Brazilian states, with the presence of vesicles that evolve to pustules and crusts, especially on the hands, arms and face, after contact with cows showing comparable lesions on the udder. In addition to the skin lesions, there have been descriptions of patients with axillary ganglionic reactions that are sometimes painful, along with fever, headache, fatigue, dehydration, anorexia, sudoresis, arthralgia and muscle pain. The condition evolves over a three to four-week period. Vulvar lesions and transmission within families have also been described. Molecular studies have shown that the poxviruses identified are genetically related to vaccinia virus samples that were used in vaccination campaigns in the past. Clinical specimens from 80 human infections were studied in the laboratory, and orthopoxvirus infections were confirmed in 68 cases. The lesions observed in these patients are presented and the implications of this zoonosis in Brazil are discussed.

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.

Background Information: VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components., Results: We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent 'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope., Conclusions: The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC(50) of 15 microM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC50>200 microM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC(50) of 45 microM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p<0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more "permissive" cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.

A new outbreak of Vaccinia virus was observed in Southwest region of São Paulo State, Brazil. The disease was observed in four small dairy farms with manual milking. Lesions were detected in cattle and in humans previously vaccinated and not vaccinated against smallpox. Although several reports of Vaccinia virus outbreaks have been occurring in Brazil, it was not yet reported in this region. This outbreak reinforces the viral circulation in our country. The disease in persons previously vaccinated and not vaccinated against smallpox reinforces the absence of immunity, the risk to the human health, and the need for more epidemiologic and immunologic studies.

The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an approximately 400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

The authors report three human cases of cowpox infection, among farm workers who were manually milking infected cows, in the microregion of Itajubá, Minas Gerais. The diagnostic techniques used were: isolation of samples similar to the vaccinia virus, from skin lesion secretions; polymerase chain reaction; electronic microscopy; and antibodies for Orthopoxvirus in the patients' blood.

Genetic and biochemical studies have provided evidence for an entry/fusion complex (EFC) comprised of at least eight viral proteins (A16, A21, A28, G3, G9, H2, J5, and L5) that together with an associated protein (F9) participates in entry of vaccinia virus (VACV) into cells. The genes encoding these proteins are conserved in all poxviruses, are expressed late in infection, and are components of the mature virion membrane but are not required for viral morphogenesis. In addition, all but one component has intramolecular disulfides that are formed by the poxvirus cytoplasmic redox system. The L1 protein has each of the characteristics enumerated above except that it has been reported to be essential for virus assembly. To further investigate the role of L1, we constructed a recombinant VACV (vL1Ri) that inducibly expresses L1. In the absence of inducer, L1 synthesis was repressed and vL1Ri was unable to form plaques or produce infectious progeny. Unexpectedly, assembly and morphogenesis appeared normal and the noninfectious virus particles were indistinguishable from wild-type VACV as determined by transmission electron microscopy and analysis of the component polypeptides. Notably, the L1-deficient virions were able to attach to cells but the cores failed to penetrate into the cytoplasm. In addition, cells infected with vL1Ri in the absence of inducer did not form syncytia following brief low-pH treatment even though extracellular virus was produced. Coimmunoprecipitation experiments demonstrated that L1 interacted with the EFC and indirectly with F9, suggesting that L1 is an additional component of the viral entry apparatus.

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be "soldered" by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.

Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25 kDa VV A12L late gene product (p17K) is proteolytically maturated at the conserved Ala-Gly-Ala motif. However, the association of the precursor and its cleavage product with the core of mature virion suggests that both of the A12L proteins may be required for virus assembly. Here, in order to test the requirement of the A12L protein and its proteolysis in viral replication, a conditional lethal mutant virus (vvtetOA12L) was constructed to regulate A12L expression by the presence or absence of an inducer, tetracycline. In the absence of tetracycline, replication of vvtetOA12L was inhibited by 80% and this inhibition could be overcome by transient expression of the wild-type copy of the A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the transfected A12L gene to rescue, indicating that A12L proteolysis plays an important role in viral replication. Electron microscopy analysis of the A12L deficient virus demonstrated the aberrant virus particles, which were displayed by the AG/A site mutation. Thus, we concluded that the not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition.