Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG(-) sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.     

The envelope G3L protein is essential for entry of vaccinia virus into host cells

The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-beta-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L(+)) or lacking (G3L(-)) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L(+) and G3L(-) IMV bound to HeLa cells; however, G3L(-) IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.     

Assembly of vaccinia virus revisited: de novo membrane synthesis or acquisition from the host?

In 1968 it was proposed that the first membrane structures that assemble in vaccinia virus-infected cells, the crescents, are formed by a unique viral mechanism in which a single membrane bilayer is synthesized de novo. 25 years later it was suggested that the vaccinia membranes are derived from an organelle that is part of the host cell's secretory pathway, the intermediate compartment (IC), and that the viral crescents are made of two tightly apposed membranes rather than a single bilayer. Several independent studies have subsequently shown that membrane proteins of the intracellular mature virus (IMV) insert co-translationally into endoplasmic reticulum (ER) membranes, and are targeted to and retained in the IC, the compartment from which the virus acquires its membranes. Furthermore, a recent study on the entry of both the IMV and extracellular enveloped virus (EEV) suggests that these viruses do not enter by a simple fusion mechanism, consistent with the idea that both are surrounded by more than one lipid bilayer.     

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.     

Visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions

Previous work indicated that vaccinia intracellular mature virus (IMV) utilizes microtubules to move from the viral factory to the site of intracellular envelopment and that expression of the viral A27 protein is required for this transport. To investigate further the role of A27 in IMV intracellular transport, a recombinant vaccinia virus was constructed that had the A27L gene deleted and expressed a yellow fluorescent protein (YFP)-A4 chimera in place of the normal A4 protein. The resulting recombinant, vYFP-A4/DeltaA27, produced relatively normal quantities of virus in a one-step growth curve but had a small plaque phenotype. Subsequent experiments demonstrated that vYFP-A4/DeltaA27 was severely defective in envelope virus production. Despite the absence of A27, live digital video fluorescent microscopy visualized YFP-labeled IMV movement in cells infected with the recombinant. Virion movement approached 3 mum/s and was sensitive to the microtubule depolymerizing drug nocodazole. In addition, IMV could be discerned transiting away from and back towards viral factories. Immunofluorescent staining determined that the distance traveled by A27-deficient virions was sufficient for transport to the site of envelopment. These results indicate that IMVs are capable of bidirectional movement on microtubules, suggesting that they are able to interact with both kinesin and dynein microtubule motors in the absence of A27 and that the distance traveled is sufficient to deliver IMV to the site of wrapping.     

The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle.

Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demonstrates a protein with an estimated molecular mass of 59 kDa, which is apparently restricted to the intracellular mature virion (IMV) form. Western blotting (immunoblotting) and immunoprecipitation procedures identify the protein as the vaccinia virus 4c protein, which facilitates occlusion of poxvirus particles within cowpox cytoplasmic inclusions. Western blotting procedures also identify the truncated A-type inclusion protein of vaccinia virus as a specific marker for IMV particles. Kinetic analyses of virion maturation and 4c production suggest that peak enveloped virion production occurs before peak IMV production in the virus replication cycle and that 4c production is concomitant with maturation of IMV. The implications for a distinct and evolutionarily conserved function of IMV in viral pathogenesis are discussed.     

Vaccinia virus WR53.5/F14.5 protein is a new component of intracellular mature virus and is important for calcium-independent cell adhesion and vaccinia virus virulence in mice

The vaccinia virus WR53.5L/F14.5L gene encodes a small conserved protein that was not detected previously. However, additional proteomic analyses of different vaccinia virus isolates and strains revealed that the WR53.5 protein was incorporated into intracellular mature virus (IMV). The WR53.5 protein contains a putative N-terminal transmembrane region and a short C-terminal region. Protease digestion removed the C terminus of WR53.5 protein from IMV particles, suggesting a similar topology to that of the IMV type II transmembrane protein. We generated a recombinant vaccinia virus, vi53.5L, that expressed WR53.5 protein under isopropyl-beta-d-thiogalactopyranoside (IPTG) regulation and found that the vaccinia virus life cycle proceeded normally with or without IPTG, suggesting that WR53.5 protein is not essential for vaccinia virus growth in cell cultures. Interestingly, the C-terminal region of WR53.5 protein was exposed on the cell surface of infected cells and mediated calcium-independent cell adhesion. Finally, viruses with inactivated WR53.5L gene expression exhibited reduced virulence in mice when animals were inoculated intranasally, demonstrating that WR53.5 protein was required for virus virulence in vivo. In summary, we identified a new vaccinia IMV envelope protein, WR53.5, that mediates cell adhesion and is important for virus virulence in vivo.     

A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus.

We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.     

A vaccinia virus core protein, p39, is membrane associated.

We describe herein the characterization of p39, the product of the A4L gene of vaccinia virus. By immunolabelling of thawed cryosections from infected HeLa cells, we show that this protein is initially located in the central region, or viroplasm, of the viral factories, as well as in the immature virions, with very small amounts of labelling observed on the surrounding membranes. The localization of p39 changes dramatically during the transition of the immature virion to the intracellular mature virus (IMV), coincident with the appearance of the core structure in the center of the IMV, with p39 located between this core and the surrounding membranes. Complementary biochemical data, such as partitioning into the Triton X-114 detergent phase and stripping of the viral membranes with Nonidet P-40 and dithiothreitol, suggest that p39 is associated with the innermost of the two membranes surrounding the core. Sodium carbonate treatment also indicates that p39 is associated with membranes, even at the early stages of viral assembly. However, following in vitro translation of p39 in the presence of microsomal membranes, we failed to detect any association of the independently expressed protein with membranes. We also failed to detect any posttranslational acylation of p39 with myristate or palmitate, suggesting that p39 does not achieve its membrane association through lipid anchors. Therefore, p39 is most likely membrane associated through an interaction with an integral membrane protein(s) present in the innermost of the two membranes surrounding the IMV. These data, together with our recent data showing that p39 colocalizes with the spike-like protrusions on the IMV core (N. Roos, M. Cyrklaff, S. Cudmore, R. Blasco, J. Krijnse-Locker, and G. Griffiths, EMBO J. 15:2343-2355, 1996), suggest that p39 may form part of this spike and that it possibly functions as a matrix-like linker protein between the core and the innermost of the two membranes surrounding the IMV.     

Hsp90 regulates activation of interferon regulatory factor 3 and TBK-1 stabilization in Sendai virus-infected cells

Interferon regulatory factor 3 (IRF3) plays a crucial role in mediating cellular responses to virus intrusion. The protein kinase TBK1 is a key regulator inducing phosphorylation of IRF3. The regulatory mechanisms during IRF3 activation remain poorly characterized. In the present study, we have identified by yeast two-hybrid approach a specific interaction between IRF3 and chaperone heat-shock protein of 90 kDa (Hsp90). The C-terminal truncation mutant of Hsp90 is a strong dominant-negative inhibitor of IRF3 activation. Knockdown of endogenous Hsp90 by RNA interference attenuates IRF3 activation and its target gene expressions. Alternatively, Hsp90-specific inhibitor geldanamycin (GA) dramatically reduces expression of IRF3-regulated interferon-stimulated genes and abolishes the cytoplasm-to-nucleus translocation and DNA binding activity of IRF3 in Sendai virus-infected cells. Significantly, virus-induced IRF3 phosphorylation is blocked by GA, whereas GA does not affect the protein level of IRF3. In addition, TBK1 is found to be a client protein of Hsp90 in vivo. Treatment of 293 cells with GA interferes with the interaction of TBK1 and Hsp90, resulting in TBK1 destabilization and its subsequent proteasome-mediated degradation. Besides maintaining stability of TBK1, Hsp90 also forms a novel complex with TBK1 and IRF3, which brings TBK1 and IRF3 dynamically into proximity and facilitates signal transduction from TBK1 to IRF3. Our study uncovers an essential role of Hsp90 in the virus-induced activation of IRF3.     

Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The cytoplasmic assembly of vaccinia virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical immature virus (IV) that matures into the intracellular mature virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia virus. p32 was associated with vaccinia virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.     

Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy

Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.     

A vaccinia virus lacking A10L: viral core proteins accumulate on structures derived from the endoplasmic reticulum

The assembly of the intracellular mature virus (IMV) of vaccinia virus (VV), the prototype member of the poxviridae, is poorly understood and controversial. We have previously proposed that the IMV is composed of a continuous double-membraned cisterna derived from the smooth ER, whereby the genome-containing core is enwrapped by a part of this cisterna. In the present study we characterize a mutant virus in which the synthesis of the major core protein A10L can be conditionally expressed. Without A10L, IMVs are not made; immature viruses (IVs) and regularly stacked membrane structures that contain viral DNA, accumulate instead. By immunolabelling of thawed cryo-sections these stacks contain most of the viral core proteins and low levels of viral membrane proteins. Importantly, the stacked membranes could be labelled with antibodies to an ER marker protein, implying that they are derived from this cellular compartment. By electron tomography (ET) on semi-thin cryo-sections we show that the membranes of the stacks are continuous with the membranes of the IVs. Direct continuities with ER cisternae, to which the stacks are tightly apposed, were, however, not unequivocally seen. Finally, ET revealed how the IV membranes separated to become two-membrane profiles. Taken together, this study shows that VV core proteins and the viral DNA can coassemble onto ER-derived membranes that are continuous with the membranes of the IVs.     

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.     

Structure and assembly of intracellular mature vaccinia virus: thin-section analyses

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.     

Vaccinia virus J1R protein: a viral membrane protein that is essential for virion morphogenesis

Vaccinia virus, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-beta-D-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.     

Vaccinia virus morphogenesis and dissemination

Vaccinia virus is the smallpox vaccine. It is the most intensively studied poxvirus, and its study has provided important insights about virus replication in general and the interactions of viruses with the host cell and immune system. Here, the entry, morphogenesis and dissemination of vaccinia virus are considered. These processes are complicated by the existence of two infectious vaccinia virus particles, called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). The IMV particle is surrounded by one membrane, and the EEV particle comprises an IMV particle enclosed within a second lipid membrane containing several viral antigens. Consequently, these virions have different biological properties and play different roles in the virus life cycle.