Remodeling of the Core Leads HIV-1 Preintegration Complex into the Nucleus of Human Lymphocytes.

Retroviral replication proceeds through obligate integration of the viral DNA into the host genome. In particular, for the HIV-1 genome to enter the nucleus, it must be led through the nuclear pore complex (NPC). During the HIV-1 cytoplasmic journey, the viral core acts as a shell to protect the viral genetic material from antiviral sensors and ensure an adequate environment for reverse transcription. However, the relatively narrow size of the nuclear pore channel requires that the HIV-1 core is reshaped into a structure that fits the pore. On the other hand, the organization of the viral CA proteins that remain associated with the preintegration complex (PIC) during and after nuclear translocation is still enigmatic. In this study, we analyzed the progressive organizational changes of viral CA proteins within the cytoplasm and the nucleus by immunogold labeling. Furthermore, we set up a novel technology, HIV-1 ANCHOR, which enables the specific detection of the retrotranscribed DNA by fluorescence microscopy, thereby offering the opportunity to uncover the architecture of the potential HIV-1 PIC. Thus, we combined the immunoelectron microscopy and ANCHOR technologies to reveal the presence of DNA- and CA-positive complexes by correlated light and electron microscopy (CLEM). During and after nuclear translocation, HIV-1 appears as a complex of viral DNA decorated by multiple viral CA proteins remodeled in a pearl necklace-like shape. Thus, we could describe how CA proteins are reshaped around the viral DNA to permit the entrance of the HIV-1 in the nucleus. This particular CA protein complex composed of the integrase and the retrotranscribed DNA leads the HIV-1 genome inside the host nucleus. Our findings contribute to the understanding of the early steps of HIV-1 infection and provide new insights into the organization of HIV-1 CA proteins during and after viral nuclear entry. Of note, we are now able to visualize the viral DNA in viral complexes, opening up new perspectives for future studies on virus's fate in the cell nucleus.IMPORTANCE How the reverse-transcribed genome reaches the host nucleus remains a main open question related to the infectious cycle of HIV-1. The HIV-1 core has a size of ∼100 nm, largely exceeding that of the NPC channel (∼39 nm). Thus, a rearrangement of the viral CA protein organization is required to achieve an effective nuclear translocation. The mechanism of this process remains undefined due to the lack of a technology capable of visualizing potential CA subcomplexes in association with the viral DNA in the nucleus of HIV-1-infected cells. By the means of state-of-the-art technologies (HIV-1 ANCHOR system combined with CLEM), our study shows that remodeled viral complexes retain multiple CA proteins but not an intact core or only a single CA monomer. These viral CA complexes associated with the retrotranscribed DNA can be observed inside the nucleus, and they represent a potential PIC. Thus, our study shed light on critical early steps characterizing HIV-1 infection, thereby revealing novel, therapeutically exploitable points of intervention. Furthermore, we developed and provided a powerful tool enabling direct, specific, and high-resolution visualization of intracellular and intranuclear HIV-1 subviral structures.

The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution.

Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.

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.

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network.

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.

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.

African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum.

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ∼5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.

Integrated electron microscopy: super-duper resolution.

Since its inception, electron microscopy (EM) has revealed that cellular membranes are organized into structurally distinct subdomains, created by localized protein and lipid assemblies to perform specific complex cellular functions. Caveolae are membrane subdomains that function as signaling platforms, endocytic carriers, sensors of membrane tension, and mechanical stress, as well as in lipid homeostasis. They were first discovered almost 60 years ago by pioneering electron microscopists. While new and exciting developments in SUPER-resolution fluorescent light microscopy facilitate studies of the spatial organization of fluorescently labeled protein components, these techniques cannot reveal the underlying cellular structures. Thus, equally exciting are developments in EM: genetically encoded probes for protein localization at sub-10 nm resolution, more powerful instruments that allow imaging of larger cell volumes, and computational methods for reconstructing three-dimensional images. Used in combination, as done by Ludwig et al. in the current issue of PLOS Biology, these tools reveal high-resolution insights into the composition and organization of the caveolae coat and the formation of these specialized structures. Together, these advances are contributing to a resurgence in EM.

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA.

Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network-associated Lck signaling.

The Nef protein of HIV-1 facilitates viral replication and disease progression in vivo. Nef disturbs the organization of immunological synapses between infected CD4(+) T lymphocytes and antigen-presenting B-lymphocytes to interfere with TCR proximal signaling. Paradoxically, Nef enhances distal TCR signaling in infected CD4(+) T lymphocytes, an effect thought to be involved in its role in AIDS pathogenesis. Using quantitative confocal microscopy and cell fractionation of Nef-expressing cells and HIV-1-infected primary human T lymphocytes, we found that Nef induces intracellular compartmentalization of TCR signaling to adjust TCR responses to antigenic stimulation. Nef reroutes kinase-active pools of the TCR signaling master switch Lck away from the plasma membrane (PM) to the trans-Golgi network (TGN), thereby preventing the recruitment of active Lck to the immunological synapse after TCR engagement and limiting signal initiation at the PM. Instead, Nef triggers Lck-dependent activation of TGN-associated Ras-Erk signaling to promote the production of the T lymphocyte survival factor IL-2 and to enhance virus spread. Overexpression of the Lck PM transporter Unc119 restores Nef-induced subversions of Lck trafficking and TCR signaling. Nef therefore hijacks Lck sorting to selectively activate TGN-associated arms of compartmentalized TCR signaling. By tailoring T-lymphocyte responses to antigenic stimulation, Nef optimizes the environment for HIV-1 replication.

Poxvirus membrane biogenesis: rupture not disruption.

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.

Open membranes are the precursors for assembly of large DNA viruses.

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.

Presence of Intact Hepatitis B Virions in Exosomes.

BACKGROUND & AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells. METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells. RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na+-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells. CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome.

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Vaccinia virus H7-protein is required for the organization of the viral scaffold protein into hexamers.

Viruses of the giant virus family are characterized by a structurally conserved scaffold-capsid protein that shapes the icosahedral virion. The vaccinia virus (VACV) scaffold protein D13, however, transiently shapes the newly assembled viral membrane in to a sphere and is absent from the mature brick-shaped virion. In infected cells D13, a 62 kDa polypeptide, forms trimers that arrange in hexamers and a honey-comb like lattice. Membrane association of the D13-lattice may be mediated by A17, an abundant 21 kDa viral membrane protein. Whether membrane binding mediates the formation of the honey-comb lattice or if other factors are involved, remains elusive. Here we show that H7, a 17 kDa protein conserved among poxviruses, mediates proper formation of D13-hexamers, and hence the honey comb lattice and spherical immature virus. Without H7 synthesis D13 trimers assemble into a large 3D network rather than the typical well organized scaffold layer observed in wild-type infection, composed of short D13 tubes of discrete length that are tightly associated with the endoplasmic reticulum (ER). The data show an unexpected role for H7 in D13 organization and imply that formation of the honey-comb, hexagonal, lattice is essential for VACV membrane assembly and production of infectious progeny. The data are discussed with respect to scaffold proteins of other giant viruses.

CD317/tetherin is enriched in the HIV-1 envelope and downregulated from the plasma membrane upon virus infection.

CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

Vaccinia virus lacking A17 induces complex membrane structures composed of open membrane sheets.

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640-4648, 1). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein.