The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion

Abstract

The L5R gene of vaccinia virus is conserved among all sequenced members of the Poxviridae but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.

FIG. 1.

L5 is conserved among members of the Poxviridae family. (A) Multiple sequence alignment of L5 orthologs, comprising a representative sequence from each genus of the Chordopoxvirinae and two available sequences from Entomopoxvirinae. Invariant cysteines are identified by white text on a black background; other invariant or similar residues in all aligned sequences are identified by shading. Abbreviations: VAC, vaccinia virus (Orthopoxvirus); MYX, myxomavirus (Leporipoxvirus); LSD, lumpy skin disease virus (Capripoxvirus); YMT, Yaba monkey tumor virus (Yatapoxvirus); SWV, swinepox virus (Suipoxvirus); ORF, ORF virus (Parapoxvirus), MOC, molluscum contagiosum virus (Molluscipoxvirus); FWP, fowlpox virus (Avipoxvirus); AMV, Amsacta moorei entomopoxvirus (Entomopoxvirus B); MSV, Melanoplus sanguinipes entomopoxvirus (Entomopoxvirus B).

FIG. 2.

L5 is synthesized with the kinetics of a typical late protein and contains an intramolecular disulfide bond formed by the vaccinia virus-encoded redox pathway. (A) Kinetics of L5 protein accumulation. BS-C-1 cells were mock infected (M) or infected with 5 PFU of vV5-L5 in the absence or presence of AraC and disrupted in SDS-PAGE loading buffer containing 20 mM N-ethylmaleimide at the indicated times postinfection. Cell lysates were subject to SDS-PAGE and Western blot analysis using an anti-V5 monoclonal antibody. (B) Evidence for an intramolecular disulfide bond. BS-C-1 cells were infected with 5 PFU per cell of vV5-L5 and incubated for 18 h. Cells were disrupted in SDS-PAGE loading buffer containing N-ethylmaleimide (NEM) or AMS. Alternatively, cells were treated with the reducing agent Tris-(2-carboxyethyl)phosphine (TCEP) prior to alkylation with either N-ethylmaleimide or AMS. Cell lysates were subjected to SDS-PAGE and Western blotting using an anti-V5 monoclonal antibody. (C) Disulfide bond formation requires the vaccinia virus E10 protein. BS-C-1 cells were infected for 2 h with either vE10i or vT7LacOI as a control. The cells were then transfected with a plasmid containing the L5R ORF with a C-terminal V5 epitope tag under the control of the natural L5R promoter. At 18 h postinfection, cells were disrupted in SDS-PAGE loading buffer containing N-ethylmaleimide or AMS. Cell lysates were subjected to SDS-PAGE and Western blot analysis using an anti-V5 monoclonal antibody. Mass standards, shown on the vertical axis, are in kDa.

FIG. 3.

L5 is an integral membrane protein exposed on the surface of intracellular mature virions. (A) Purified vaccinia virus WR or vV5-L5 intracellular mature virions were suspended in Tris buffer, pH 7.4, and incubated at 37°C for 30 min, with or without the addition of 1% NP-40 or 50 mM dithiothreitol (DTT) as indicated. After 30 min, virus suspensions were centrifuged at 20,000 × g for 30 min at 4°C and separated into pellet (P) and supernatant (S) fractions. The separate fractions were solubilized in SDS-PAGE loading buffer and the resulting lysates were subject to SDS-PAGE and Western blotting using anti-V5 and anti-L1 antibodies. (B) Purified vV5-L5 intracellular mature virions were treated with trypsin, NP-40, or NP-40 plus trypsin. Pellet and supernatant fractions were analyzed by SDS-PAGE. Mass markers, shown on the vertical axis, are in kDa. (C) Biotinylation of surface proteins. Purified intact or NP-40-disrupted vV5-L5 intracellular mature virions were treated or mock treated with sulfo-NHS-SS-biotin as previously described (40). After SDS dissociation, the proteins were incubated with neutravidin beads and bound (B) and unbound (U) proteins were analyzed by SDS-PAGE followed by Western blotting with antibodies to the V5 epitope tag to reveal L5 or to the D8 surface membrane protein or the A10 core protein. The analysis of biotinylated D8 and A10 was presented previously (40).

FIG. 4.

Conditional lethal phenotype of a recombinant vaccinia virus with an inducible L5R gene. (A) Schematic of portions of the vV5-L5i genome. Abbreviations: lacO, E. coli lac operator; P₁₁, vaccinia virus late promoter; P_7.5, vaccinia virus early/late promoter; lacI, E. coli lac repressor gene; P_T7, bacteriophage T7 promoter. (B) Plaque formation. BS-C-1 monolayers were infected with vV5-L5i in the presence (+) or absence (−) of IPTG. At 24 h postinfection, monolayers were examined by fluorescence microscopy for the presence of EGFP. Arrows point to fluorescent cells. At 48 h, monolayers were fixed and stained with crystal violet. (C) Single-step virus yields. BS-C-1 monolayers were infected with 5 PFU per cell of the indicated virus in the presence (+) or absence (−) of IPTG. The cells were harvested at the indicated hours postinfection, and virus titers were determined by plaque assay in the presence of 50 μM IPTG. The experiment was performed in duplicate, and the data points represent the mean ± standard error of the mean. At some points, the error bars are too close to resolve.

FIG. 5.

vV5-L5i produces enveloped virions with actin tails in the absence of IPTG. HeLa cell monolayers were infected with 5 PFU per cell of vV5-L5i and incubated in the absence (−) or presence (+) of IPTG for 20 h. The infected cells were fixed with 3% paraformaldehyde and quenched with 2% glycine. Cell surface cell-associated extracellular enveloped virions were labeled with anti-B5R monoclonal antibody and Cy5-conjugated goat anti-rat secondary antibody. Following this, cells were permeabilized by the addition of 0.1% Triton X-100 and stained with DAPI to visualize nuclei and Alexa Fluor 568-phalloidin to visualize filamentous actin. Arrows point to representative cell-associated extracellular enveloped virions at the tips of actin tails.

FIG.6.

Morphology and polypeptide composition of virions lacking or containing L5. (A) Electron microscopy of purified virions. Intracellular mature virions were purified by sucrose gradient sedimentation from cells infected with vV5-L5i in the presence (+ L5) or absence (−L5) of IPTG. Virions were deposited on grids, washed with water, and stained with 7% uranyl acetate in 50% ethanol for 30 seconds. (B) SDS-PAGE. Equivalent amounts of sucrose gradient-purified -L5, +L5, and vaccinia virus WR virions were solubilized in SDS-PAGE loading buffer and subjected to SDS-PAGE and silver staining. Mass markers are indicated in kDa on the left. (C) Western blotting. SDS-PAGE was performed as above, and the proteins were transferred to a membrane and probed with antibodies to the A28, A21, and L5 proteins.

FIG.7.

Cells infected with -L5 virions exhibit reduced early-gene expression. (A) Northern blot analysis. BS-C-1 cell monolayers were mock infected or infected with 5 PFU per cell of +L5 virions or the corresponding OD₂₆₀ of -L5 virions. Total RNA was harvested 3 h postinfection and subjected to Northern blot analysis with [α-³²P]dCTP-labeled double-stranded DNA probes specific for the vaccinia virus growth factor (C11R), DNA polymerase (E9L), or cellular actin transcripts. (B) In vitro RNA synthesis. Purified +L5 and -L5 virions were permeabilized with NP-40 and incubated with ribonucleoside triphosphates and [α-³²P]UTP. The incorporation of [α-³²P]UMP into trichloroacetic acid-insoluble material was determined by scintillation counting and is expressed as counts per minute (cpm).

FIG. 8.

Virions lacking L5 are unable to release their cores into the cytosol. Replicate HeLa cell monolayers were inoculated with 20 PFU per cell of purified +L5 virions or the corresponding OD₂₆₀ of -L5 virions at 4°C for 1 h. The cells were washed extensively and fixed or incubated for a further 2 h at 37°C in the presence of cycloheximide before fixation. Autofluorescence was quenched with 2% glycine, and cells were permeabilized with 0.1% Triton X-100. Cells were labeled with mouse anti-L1 antibody to detect intracellular mature virions on the cell surface and rabbit anti-A4 antibody to detect the A4 core protein in the cytoplasm, followed by fluorescein isothiocyanate-conjugated goat anti-mouse (green) and rhodamine red-X-conjugated goat anti-rabbit (red) antibody, respectively, as described (40). DNA was visualized by staining with DAPI. Immunolabeled cells were visualized by confocal microscopy as a sequence of optical sections, which are displayed as maximum-intensity projections.

FIG. 9.

Virions lacking L5 are unable to mediate low-pH-triggered cell-cell fusion. (A) Fusion from without. BS-C-1 cells were inoculated with either 200 PFU per cell of purified +L5 virions or the corresponding OD₂₆₀ of -L5 virions at 4°C for 1 h. The cells were immersed in pH 5.3 or pH 7.4 buffer at 37°C. After 3 min, the buffers were replaced with culture medium containing 300 μg of cycloheximide per ml and incubated for 3 h at 37°C. The cells were then fixed and stained with Alexa Fluor 568-phalloidin and DAPI to display actin filaments and DNA, respectively. Confocal microscopy images are shown. The extensive actin rearrangement helps to visualize the syncytia. (B) Fusion from within. BS-C-1 cell monolayers were infected with 5 PFU per cell of vV5-L5i in the presence or absence of 50 μM IPTG for 18 h and transiently treated with either pH 5.3 or pH 7.4 buffer as above before the buffer was replaced with culture medium. Cells were stained with DAPI and visualized by phase and fluorescence microscopy. vV5-L5i constitutively synthesizes EGFP in the presence or absence of IPTG, and its cytoplasmic distribution served as an indicator of cell-cell fusion.