The Role of the Transmembrane and of the Intraviral Domain of Glycoproteins in Membrane Fusion of Enveloped Viruses

Fusion of enveloped viruses with their target membrane is mediated by viral integral glycoproteins. A conformational change of their ectodomain triggers membrane fusion. Several studies suggest that an extended, triple-stranded rod-shaped -helical coiled coil resembles a common structural and functional motif of the ectodomain of fusion proteins. From that, it is believed that essential features of the fusion process are conserved among the various enveloped viruses. However, this has not been established so far for the highly conserved transmembrane and intraviral sequences of fusion proteins. The article will focus on the role of both sequences in the fusion process. Recent studies from various enveloped viruses strongly imply that a transmembrane domain with a minimum length is required for later steps of membrane fusion, i.e., the formation and enlargement of the aqueous fusion pore. Although no specific sequence of the TM is necessary for pore formation, distinct properties and motifs of the domain may be obligatory to ascertain full fusion activity. However, with some exceptions, the intraviral domain seems to be not required for fusion activity of viral fusion proteins.     

Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells.

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.     

Inactivation of laboratory animal RNA-viruses by physicochemical treatment

Eight commonly used chemical disinfectants and physical treatments (UV irradiation and heating) were applied to both enveloped RNA viruses (Sendai virus, canine distemper virus) and unenveloped RNA viruses (Theiler's murine encephalomyelitis virus, reo virus type 3) to inactivate infectious virus particles. According to the results, alcohols (70% ethanol, 50% isopropanol), formaldehyde (2% formalin), halogen compounds (52ppm iodophor, 100ppm sodium hypochlorite), quaternary ammonium chloride (0.05% benzalkonium chloride) and 1% saponated cresol showed virucidal effects giving more than 99.95% reduction in the infectivity of virus samples of Sendai virus and canine distemper after 10 minutes exposure. There was no significant difference in the effects on the two enveloped RNA viruses. The susceptibility of unenveloped RNA viruses to chemical disinfectants and physical treatments differed greatly from the enveloped viruses. The two unenveloped viruses showed distinct resistance to 50% isopropanol, 2% formalin, 1% saponated cresol and to physical treatments (heating at 45, 56, 60 degrees C, and UV irradiation). These results indicate that using physicochemical methods to inactivate RNA viruses in laboratory animal facilities should be considered in accordance with the characteristics of the target virus. For practical purposes in disinfecting enveloped RNA viruses, 70% ethanol, 0.05% quaternary ammonium chloride and 1% saponated cresol diluted in hot water (greater than 60 degrees C) are considered as effective as UV irradiation. For unenveloped RNA viruses, halogen compounds, more than 1,000 ppm sodium hypochlorite or 260 ppm iodophor are recommended over a period of 10 minutes for disinfecting particles, although these compounds result in an oxidation problem with many metals.     

The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.     

Virus Maturation by Budding

Enveloped viruses mature by budding at cellular membranes. It has been generally thought that this process is driven by interactions between the viral transmembrane proteins and the internal virion components (core, capsid, or nucleocapsid). This model was particularly applicable to alphaviruses, which require both spike proteins and a nucleocapsid for budding. However, genetic studies have clearly shown that the retrovirus core protein, i.e., the Gag protein, is able to form enveloped particles by itself. Also, budding of negative-strand RNA viruses (rhabdoviruses, orthomyxoviruses, and paramyxoviruses) seems to be accomplished mainly by internal components, most probably the matrix protein, since the spike proteins are not absolutely required for budding of these viruses either. In contrast, budding of coronavirus particles can occur in the absence of the nucleocapsid and appears to require two membrane proteins only. Biochemical and structural data suggest that the proteins, which play a key role in budding, drive this process by forming a three-dimensional (cage-like) protein lattice at the surface of or within the membrane. Similarly, recent electron microscopic studies revealed that the alphavirus spike proteins are also engaged in extensive lateral interactions, forming a dense protein shell at the outer surface of the viral envelope. On the basis of these data, we propose that the budding of enveloped viruses in general is governed by lateral interactions between peripheral or integral membrane proteins. This new concept also provides answers to the question of how viral and cellular membrane proteins are sorted during budding. In addition, it has implications for the mechanism by which the virion is uncoated during virus entry.     

Adenovirus protein VI mediates membrane disruption following capsid disassembly

In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic alpha-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.     

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.     

The Envelope Protein Encoded by the A33R Gene Is Required for Formation of Actin-Containing Microvilli and Efficient Cell-to-Cell Spread of Vaccinia Virus

The vaccinia virus (VV) A33R gene encodes a highly conserved 23- to 28-kDa glycoprotein that is specifically incorporated into the viral outer envelope. The protein is expressed early and late after infection, consistent with putative early and late promoter sequences. To determine the role of the protein, two inducible A33R mutants were constructed, one with the late promoter and one with the early and late A33R promoter elements. Decreased A33R expression was associated with small plaques that formed comets in liquid medium. Using both an antibiotic resistance gene and a color marker, an A33R deletion mutant, vA33Δ, was isolated, indicating that the A33R gene is not essential for VV replication. The plaques formed by vA33Δ, however, were tiny, indicating that the A33R protein is necessary for efficient cell-to-cell spread. Rescue of the large-plaque phenotype was achieved by inserting a new copy of the A33R gene into the thymidine kinase locus, confirming the specific genetic basis of the phenotype. Although there was a reduction in intracellular virus formed in cells infected with vA33Δ, the amount of infectious virus in the medium was increased. The virus particles in the medium had the buoyant density of extracellular enveloped viruses (EEV). Additionally, amounts of vA33Δ cell-associated extracellular enveloped viruses (CEV) were found to be normal. Immunogold electron microscopy of cells infected with vA33Δ demonstrated the presence of the expected F13L and B5R proteins in wrapping membranes and EEV; however, fully wrapped vA33Δ intracellular enveloped viruses (IEV) were rare compared to partially wrapped particles. Specialized actin tails that propel IEV particles to the periphery and virus-tipped microvilli (both common in wild-type-infected cells) were absent in cells infected with vA33Δ. This is the first deletion mutant in a VV envelope gene that produces at least normal amounts of fully infectious EEV and CEV and yet has a small-plaque phenotype. These data support a new model for VV spread, emphasizing the importance of virus-tipped actin tails.     

Early molecular events during the interaction of enveloped riboviruses with cells. II. A kinetic study.

A kinetic model was constructed and partly solved to describe the migration of the fluorescence label 1,6-diphenylhexatriene (DPH) in both directions when enveloped viruses, labelled with DPH in their envelopes are in contact with unlabelled cells or cell labelled in their membranes are in contact with unlabelled enveloped viruses. The central assumption is that two types of receptor sites exist on the cell surface, i.e., physical adsorption sites (P-sites), available to all the viruses studied in these papers and binding sites (B-sites) available only to the viruses which penetrate into the specific cells. The differential equations for the label migration, for different values of the ratio number of viruses number of sites were numerically solved, assuming different fractions of P- and B-sites. The equations also describe, appropriately the mechanism of rapid label migration in the system and substantiate the magnitude "time of residence" of the nonpenetrating viruses adsorbed on the cell surface. The resulting curves match satisfactorily those for the label release by the viruses and account well for the steady state values of the kinetics of label migration in the virus-cell system.     

Sequences determining the pH dependence of viral entry are distinct from the host range-determining region of the murine ecotropic and amphotropic retrovirus envelope proteins.

The entry of ecotropic and amphotropic murine leukemia retroviruses (MuLV) into cells was investigated by using viral vector particles carrying chimeric amphotropic-ecotropic envelope glycoproteins on their surface. Chimeras were made by joining, at or near the polyproline hinge, the N-terminal portion of the amphotropic (4070A) gp70 onto the C-terminal portion of the ecotropic (Moloney) gp70 and p15E (constructs AE2 and AE4) or vice versa (AE12). Transduction efficiency of the constructs was tested on target cells that either have only ecotropic receptors (CHO-2 and CHO-11 cells), only amphotropic receptors (mink lung fibroblasts and Cos 1 cells), or both types of receptors (NIH 3T3 cells). The assay made use of the fact that the mechanism for viral entry of ecotropic viruses is pH dependent while that of amphotropic viruses is pH independent. Treatment of target cells with NH4Cl, which prevents the reduction of pH within endosomes, reduced the titers of viral particles bearing the C-terminal moiety from the ecotropic envelope but did not reduce the titers of particles which had a C-terminal moiety from the amphotropic envelope. In addition, in contrast to other low-pH-dependent enveloped viruses, brief acid treatment did not allow surface-bound viruses to bypass the NH4Cl block. The results indicate that the pH dependence of viral entry is a property of the sequences C terminal to the polyproline hinge.     

Physical and Biochemical Properties of Progressive Pneumonia Virus

The physical and biochemical characteristics of progressive pneumonia virus were found to be remarkably similar to those of the ribonucleic acid (RNA) tumor viruses. Significant findings included the presence of a 60 to 70S RNA genome, RNA-dependent deoxyribonucleic acid polymerase activity, and common morphological properties. This information correlates with previously reported biological observations and supports the provisional inclusion of enveloped RNA-containing "slow viruses" within the RNA tumor virus group.     

Membrane remodeling by the double-barrel scaffolding protein of poxvirus

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.     

Effects of protein kinase C inhibitors on viral entry and infectivity.

The protein kinase C inhibitor H-7 (2-20 microM) inhibited dose-dependently the infectivity of the vesicular stomatitis virus on cultured human fibroblasts. Electron microscopy showed that H-7 inhibited the viral entry. H-7 also inhibited the infectivity of four other enveloped viruses, herpes simplex I, turkey herpes, vaccinia and Sindbis. Similar results were obtained using staurosporine (2.5 nM), tamoxifen (40 microM), phloretin (140 microM), or W-7 (40 microM). However, the infectivity of non-enveloped viruses (e.g. poliomyelitis I) was not inhibited by H-7. These results show that protein kinase C is critically involved in the infectivity of enveloped viruses, most probably at the level of viral entry (receptor-mediated endocytosis).     

The kinase inhibitor SFV785 dislocates dengue virus envelope protein from the replication complex and blocks virus assembly

Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.     

Double-labeled rabies virus: live tracking of enveloped virus transport

Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.     

Why Enveloped Viruses Need Cores—The Contribution of a Nucleocapsid Core to Viral Budding

During the lifecycle of many enveloped viruses, a nucleocapsid core buds through the cell membrane to acquire an outer envelope of lipid membrane and viral glycoproteins. However, the presence of a nucleocapsid core is not required for assembly of infectious particles. To determine the role of the nucleocapsid core, we develop a coarse-grained computational model with which we investigate budding dynamics as a function of glycoprotein and nucleocapsid interactions, as well as budding in the absence of a nucleocapsid. We find that there is a transition between glycoprotein-directed budding and nucleocapsid-directed budding that occurs above a threshold strength of nucleocapsid interactions. The simulations predict that glycoprotein-directed budding leads to significantly increased size polydispersity and particle polymorphism. This polydispersity can be explained by a theoretical model accounting for the competition between bending energy of the membrane and the glycoprotein shell. The simulations also show that the geometry of a budding particle leads to a barrier to subunit diffusion, which can result in a stalled, partially budded state. We present a phase diagram for this and other morphologies of budded particles. Comparison of these structures against experiments could establish bounds on whether budding is directed by glycoprotein or nucleocapsid interactions. Although our model is motivated by alphaviruses, we discuss implications of our results for other enveloped viruses.     

Inhibition of enveloped virus infection of cultured cells by valproic acid

Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.     

Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of rous sarcoma and Rauscher murine leukemia cells

Summary Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27–1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [³²P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [³²P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cellsin vivo is discussed.