Intracellular Enveloped Virus Research Articles

Mpox: What Practicing Gastroenterologists Need to Know

Mpox: What Practicing Gastroenterologists Need to Know

Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library.

Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction.

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress.

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Tagging of the vaccinia virus protein F13 with mCherry causes aberrant virion morphogenesis.

Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.

Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis.

ABSTRACTPoxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.IMPORTANCE Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.

Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor.

Shape and energy of a membrane bud induced by protein coats or viral protein assembly.

Intracellular transport vesicles and enveloped virus production is mediated by the polymerization of proteins that form bi-dimensional curved and rigid structures, or "coats", on a membrane. Using the classical framework of fluid membrane elasticity, we compute numerically the shape and the mechanical energy of the membrane deformation induced by a coat at different stage of growth. We furthermore derive analytical approximate expressions for the membrane shape and energy. They are found to be very accurate when compared to numerical calculations. These analytical expressions should be useful when building a relevant model of coat polymerization kinetics. We also discuss some consequences of the membrane energy features on the coat assembly process, showing that at high tension a kinetically arrested state of incomplete assembly could exist.

The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs.

Marek's Disease Virus Morphogenesis

Marek's disease virus (MDV) is a highly contagious virus that induces T-lymphoma in chicken. This viral infection still circulates in poultry flocks despite the use of vaccines. With the emergence of new virulent strains in the field over time, MDV remains a serious threat to the poultry industry. More than 40 yr after MDV identification as a herpesvirus, the visualization and purification of fully enveloped infectious particles remain a challenge for biologists. The various strategies used to detect such hidden particles by electron microscopy are reviewed herein. It is now generally accepted that the production of cell-free virions only occurs in the feather follicle epithelium and is associated with viral, cellular, or both molecular determinants expressed in this tissue. This tissue is considered the only source of efficient virus shedding into the environment and therefore the origin of successful transmission in birds. In other avian tissues or permissive cell cultures, MDV replication only leads to a very low number of intracellular enveloped virions. In the absence of detectable extracellular enveloped virions in cell culture, the nature of the transmitted infectious material and its mechanisms of spread from cell to cell remain to be deciphered. An attempt is made to bring together the current knowledge on MDV morphogenesis and spread, and new approaches that could help understand MDV morphogenesis are discussed.

Transport and stability of the vaccinia virus A34 protein is affected by the A33 protein

Vaccinia virus (VACV) has two infectious forms called intracellular mature virus and extracellular enveloped virus (EEV). Two of the seven viral proteins in the EEV outer envelope, A33 and A34, are type II membrane glycoproteins that each interact with another EEV protein called B5; however, evidence for direct A33–A34 interaction is lacking. The localization and stability of A34 is affected by B5 and here data are presented showing that A34 is also affected by A33. In the absence of A33, just as without B5, the level, localization and glycosylation profile of A34 was altered. However, the glycosylation profile of A34 without A33 is different to that observed in the absence of B5, and A34 accumulates in the Golgi apparatus rather than in the endoplasmic reticulum. Thus, A34 requires more than one other EEV protein for its processing and cellular transport.

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis

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 (p85α−/−β−/−) 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 p85α−/−β−/− 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.

An E2-F12 complex is required for intracellular enveloped virus morphogenesis during vaccinia infection

The vaccinia virus protein, F12, has been suggested to play an important role in microtubule-based transport of intracellular enveloped virus (IEV). We found that GFP-F12 is recruited to IEV moving on microtubules but is released from virus particles when they switch to actin-based motility. In the absence of F12, although the majority of IEV remain close to their peri-nuclear site of assembly, a small number of IEV still move with linear trajectories at speeds of 0.85 μm s−1, consistent with microtubule transport. Using a recombinant virus expressing GST-F12, we found that the viral protein E2 interacts directly with F12. In infected cells, GFP-E2 is observed on moving IEV as well as in the Golgi region, but is not associated with actin tails. In the absence of E2L, IEV accumulate in the peri-nuclear region and F12 is not recruited. Conversely, GFP-E2 is not observed on IEV in the absence of F12. Ultra-structural analysis of ΔE2L- and ΔF12L-infected cells reveals that loss of either protein results in defects in membrane wrapping during IEV formation. We suggest that E2 and F12 function as a complex that is necessary for IEV morphogenesis prior to their microtubule-based transport towards the plasma membrane.

Vaccinia virus protein F12 associates with intracellular enveloped virions through an interaction with A36.

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.

Activity of the Anti-Orthopoxvirus Compound ST-246 against Vaccinia, Cowpox and Camelpox Viruses in Cell Monolayers and Organotypic Raft Cultures

The potential use of variola virus as a biological weapon has renewed efforts in the development of antiviral agents against orthopoxviruses. ST-246 [4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-di oxo-4,6-ethenocycloprop [f]isoindol-2(1 H)-yl)-benzamide] is an anti-orthopoxvirus compound active against several orthopoxviruses including vaccinia virus (VV), cowpox virus (CPV), camelpox virus (CMLV), ectromelia virus (ECTV) and variola virus in cell culture. The compound has been shown to inhibit the release of extracellular virus by targeting the F13L W protein and to protect mice from W, CPV and ECTV orthopoxvirus-induced disease. The antiviral activity of ST-246 was assessed against extracellular and intracellular W, CPV and CMLV production in human embryonic lung (HEL) fibroblasts and primary human keratinocyte (PHK) cell monolayers, as well as in three-dimensional raft cultures. ST-246 inhibited preferentially the production of extracellular virus compared with intracellular virus production in HEL and PHK cells (for W) and in PHK cells (for CMLV). In organotypic epithelial raft cultures, ST-246 at 20 microg/ml inhibited extracellular W and CMLV production by 6 logs, whereas intracellular virus yield was reduced by 2 logs. In the case of CPV, both extracellular and intracellular virus production were completely inhibited by ST-246 at 20 microg/ml. Histological sections of the infected rafts, treated with increasing amounts of drug, confirmed the antiviral activity of ST-246: the epithelium was protected and there was no evidence of viral infection. Electron microscopic examination confirmed the absence of intracellular enveloped virus forms in W-, CPV- and CMLV-infected cells treated with 10 microg/ml of ST-246. These data indicate that ST-246 is a potent anti-orthopoxvirus compound; the mode of inhibition is dependent on the virus and cell type.

Morphogenesis of a Highly Replicative EGFPVP22 Recombinant Marek's Disease Virus in Cell Culture

Marek's disease virus (MDV) is an alphaherpesvirus for which infection is strictly cell associated in permissive cell culture systems. In contrast to most other alphaherpesviruses, no comprehensive ultrastructural study has been published to date describing the different stages of MDV morphogenesis. To circumvent problems linked to nonsynchronized infection and low infectivity titers, we generated a recombinant MDV expressing an enhanced green fluorescent protein fused to VP22, a major tegument protein that is not implicated in virion morphogenesis. Growth of this recombinant virus in cell culture was decreased threefold compared to that of the parental Bac20 virus, but this mutant was still highly replicative. The recombinant virus allowed us to select infected cells by cell-sorting cytometry at late stages of infection for subsequent transmission electron microscopy analysis. Under these conditions, all of the stages of assembly and virion morphogenesis could be observed except extracellular enveloped virions, even at the cell surface. We observed 10-fold fewer naked cytoplasmic capsids than nuclear capsids, and intracellular enveloped virions were very rare. The partial envelopment of capsids in the cytoplasm supports the hypothesis of the acquisition of the final envelope in this cellular compartment. We demonstrate for the first time that, compared to other alphaherpesviruses, MDV seems deficient in three crucial steps of viral morphogenesis, i.e., release from the nucleus, secondary envelopment, and the exocytosis process. The discrepancy between the efficiency with which this MDV mutant spreads in cell culture and the relatively inefficient process of its envelopment and virion release raises the question of the MDV cell-to-cell spreading mechanism.

The Release of Vaccinia Virus from Infected Cells Requires RhoA-mDia Modulation of Cortical Actin

Prior to being released from the infected cell, intracellular enveloped vaccinia virus particles are transported from their perinuclear assembly site to the plasma membrane along microtubules by the motor kinesin-1. After fusion with the plasma membrane, stimulation of actin tails beneath extracellular virus particles acts to enhance cell-to-cell virus spread. However, we lack molecular understanding of events that occur at the cell periphery just before and during the liberation of virus particles. Using live cell imaging, we show that virus particles move in the cell cortex, independently of actin tail formation. These cortical movements and the subsequent release of virus particles, which are both actin dependent, require F11L-mediated inhibition of RhoA-mDia signaling. We suggest that the exit of vaccinia virus from infected cells has strong parallels to exocytosis, as it is dependent on the assembly and organization of actin in the cell cortex.

A trans -Golgi Network Resident Protein, golgin-97, Accumulates in Viral Factories and Incorporates into Virions during Poxvirus Infection

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described here, we demonstrate that a host cell protein residing in the trans-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. Here we discuss the potential role and functions of golgin-97 in poxvirus replication and propose two working models.

Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein

Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vdeltaF12L) or A36R (vdeltaA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at approximately 60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vdeltaA36R-infected cells. Moreover, vdeltaA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vdeltaA36R IEV moved in a stop-start manner at speeds consistent with microtubular movement, although the mean length of vdeltaA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.