A28 is highly conserved in all sequenced poxvirus genomes. The ORF designated A28L in the Copenhagen strain of vaccinia virus and VACWR151 in the WR strain is predicted to encode a 16.3-kDa protein, which is conserved in all sequenced poxvirus genomes. We could find no significant sequence similarity between A28 and any nonpoxvirus protein, in spite of extensive attempts using iterative database searches with the PSI-BLAST program. All A28 orthologs contain a highly hydrophobic N-terminal region, which could function as a signal peptide or a transmembrane anchor; four conserved cysteines potentially involved in disulfide bond formation; and several invariant charged and polar residues (Fig. ). The nonhydrophobic portion of A28 was predicted to have primarily a β-sheet structure, with six putative β-strands and two flanking α-helices (Fig. ).
FIG. 1. Multiple alignment of A28 orthologs. The alignment includes one representative sequence from each genus of Chordopoxvirinae for which such information is available and the two complete Entomopoxvirinae sequences. VAC, vaccinia virus (Orthopoxvirus); MCV, (more ...) Synthesis of A28 during the vaccinia virus replicative cycle.
The start codon of the A28L ORF forms part of a TAAATG sequence, which is typical of promoters of late genes in poxviruses (5
). To monitor the synthesis of A28 during the vaccinia virus replicative cycle, we constructed a recombinant vaccinia virus in which the coding sequence for a 9-amino-acid influenza virus HA epitope was linked to the 3′ terminus of the A28L gene, which accordingly remained under the control of the native promoter (Fig. ). The GUS gene under the control of an early-late vaccinia virus promoter was coinserted into the vaccinia virus genome to allow screening for recombinant virus plaques. The recombinant virus, called vA28-HA, was clonally purified and characterized. Neither the virus yield nor the plaque size of the recombinant virus was impaired compared to wild-type virus (data not shown).
FIG. 2. Temporal synthesis of A28. (A) Construction of vA28-HA. The DNA used for the insertion of the HA tag coding sequence at the 3′ end of the A28L ORF by homologous recombination was constructed by PCR and is shown schematically. A29L and A27L are (more ...)
Cells were infected with vA28-HA in the absence or presence of cytosine arabinoside (AraC), an inhibitor of DNA replication, and the lysates were analyzed by SDS-PAGE under nonreducing conditions. A 17-kDa band was detected by sensitive Western blotting with anti-HA antibody at zero time, reflecting the presence of A28-HA in the inoculum (Fig. ), which was not seen in lysates of uninfected cells (not shown). The amount of A28-HA increased from 4 h after infection, the usual time of onset of viral late-protein synthesis. A28-HA was not elevated above the zero time background when viral DNA replication was inhibited with AraC, as expected for a late protein (Fig. ). Prominent bands of 24 and 27 kDa were also detected in cells infected with vA28-HA (Fig. ) but not in cells infected with the wild-type virus lacking the epitope tag (not shown). The 24- and 27-kDa species accumulated with the same kinetics as the 17-kDa protein during the first 7 h of infection but did not increase thereafter. Only the 17-kDa form of A28-HA was incorporated into virus particles, as can be seen in the zero time lane (Fig. ) and by analysis of purified virions (16a
). We presume that the 24- and 27-kDa proteins represent modified or tight complexes of A28-HA, and their identification by mass spectroscopy is planned.
A28-HA contains two intramolecular disulfide bonds.
A28 and all poxvirus homologs were predicted to have four conserved cysteines within the nonhydrophobic segment of the protein (Fig. ). In some cases, intramolecular disulfide-bonded proteins have a compact structure and can be resolved from their reduced forms by SDS-PAGE. We therefore compared the mobilities of A28-HA proteins from infected cells under reducing and nonreducing conditions but found no difference (data not shown). To explain this result, we considered three possibilities: (i) the cysteines of A28 are not disulfide bonded, (ii) A28 has disulfide bonds that are not reducible by the methods used, or (iii) A28 has disulfide bonds but their reduction does not alter the electrophoretic mobility under the gel conditions used. To detect free cysteines, we used AMS as an alkylating agent. For each reactive cysteine, AMS increases the mass of a protein by 0.536 kDa. Because nonalkylated proteins can become alkylated with unpolymerized acrylamide during electrophoresis, we used NEM-alkylated A28 as the control. NEM increases the mass by only 0.125 kDa per reactive cysteine. Therefore, if A28 has four reactive cysteines, there would be a mass difference of 1.6 kDa between the AMS- and NEM-alkylated proteins, which would be easily discerned by SDS-PAGE. A28-HA synthesized in vitro had free cysteines, as indicated by the clear decrease in mobility when AMS was used as an alkylating agent (Fig. ). A28-HA synthesized in infected cells exhibited no mobility change with AMS treatment (Fig. ), indicating the absence of free cysteines. However, when A28-HA from infected cells was first reduced with Tris-(2-carboxyethyl) phosphine and then alkylated with AMS, the mass of the majority of the protein increased by ~2 kDa. Therefore, the third explanation was correct: A28 from infected cells contains two reducible disulfide bonds. The same result was obtained when A28 was extracted from purified virions (data not shown).
FIG. 3. Formation of disulfide bonds in A28-HA. (A) A28-HA contains two intramolecular disulfide bonds. In vitro, A28-HA synthesized in a coupled transcription-translation recticulocyte lysate system was captured with anti-HA antibody, and aliquots were disrupted (more ...) A28 is a substrate of the viral disulfide bond formation pathway.
Previously, Senkevich et al. (19
) described a unique poxvirus-specific pathway for disulfide bond formation that operates on the cytoplasmic side of the IMV membrane, creating intramolecular disulfide bonds in at least two vaccinia virus membrane proteins. A28, with two disulfide bonds and a presumptive membrane topology, was a good candidate for another substrate of this pathway. To determine whether the viral pathway is required for the formation of disulfide bonds in A28, we transfected the plasmid encoding A28 under the control of a strong late vaccinia virus promoter into cells infected with a vaccinia virus mutant that does not express one of the three proteins comprising this pathway (E10, A2.5, or G4) in the absence of IPTG. As a control, cells were also infected with the parental virus, vT7lacOI, which expresses all three proteins in the absence of IPTG. The results of the experiments with vE10i and vG4i are shown in Fig. , and corresponding results were obtained with vA2.5i (data not shown). AMS was used to distinguish between reduced A28 with free reactive cysteines and oxidized A28 with unreactive disulfide bonds. A28 was completely reduced when expressed in cells unable to synthesize E10, A2.5, or G4 and mostly oxidized when expressed in cells making these proteins. We attribute incomplete disulfide bond formation under the latter conditions to the overexpression of A28 under the strong P11 promoter, as the same phenomenon was observed when other disulfide-bonded proteins were overexpressed (20
). Thus, the expression of E10, A2.5, and G4 was required for the formation of disulfide bonds in A28.
Localization of A28 in virus particles.
A28 was tightly associated with purified IMV and was not released by incubation in Tris (pH 7.5) buffer at 37°C for 30 min (Fig. ). The similar sizes of A28 made in vitro and in infected cells (Fig. ) suggested that the N-terminal hydrophobic sequence of A28 was not cleaved from the rest of the protein and most likely functions as a transmembrane anchor that holds the protein in the membrane of IMV. The distribution of A28 between nonionic-detergent-soluble and -insoluble fractions of purified virions was in agreement with its membrane association. At least 50% of A28 could be extracted from purified virions with NP-40 alone, and there was no difference, in this respect, between A28 and L1, a well-characterized IMV membrane protein (Fig. ). Unexpectedly, but reproducibly, less A28 was extracted when dithiothreitol was included in addition to NP-40. It might be that A28 is less soluble, interacts with insoluble proteins of the core, or is more susceptible to degradation when artificially reduced in the absence of a denaturing agent.
FIG. 4. Extraction of A28-HA from purified virions. Triplicate samples of sucrose gradient-purified virions were resuspended in Tris buffer alone (50 mM Tris, pH 7.5) or in Tris buffer containing (+) 1% NP-40 or 50 mM dithiothreitol and incubated for (more ...)
To determine the membrane orientation of the C-terminal portion of A28, purified A28-HA virions were incubated with anti-HA antibody and examined by immunoelectron microscopy. Purified E10-HA and wild-type WR virions were used as positive and negative controls, respectively. Gold particles conjugated to the secondary antibody were detected on the surfaces of both E10-HA and A28-HA virions, in contrast to the near absence of surface staining of control wild-type virions lacking an HA-tagged protein (Fig. ). The fewer gold particles detected on A28-HA virions than on E10-HA virions probably reflected the relative amounts of the two proteins in virions. We also detected less A28-HA than E10-HA by Western blotting of extracts from the same number of infected cells or the same number of purified virions (data not shown), suggesting differences in the expression levels of the two proteins. Taken together, the data suggested that A28 is anchored in the IMV membrane via its hydrophobic N terminus, with the rest of the molecule exposed on the virion surface.
FIG. 5. Immunoelectron microscopy of purified virions. Purified IMV preparations of A28-HA, E10-HA, or wild-type vaccinia virus WR were adsorbed to grids and stained with a mouse monoclonal anti-HA antibody, followed by anti-mouse antibody and protein A conjugated (more ...) A28 is essential for virus replication and plaque formation.
Because of the conservation of the A28L gene in all poxviruses, it seemed likely that its expression would be essential for virus replication in cell culture. To investigate this, we constructed the recombinant vaccinia virus vA28-HAi, which encodes the E. coli lac repressor continuously expressed by a vaccinia virus dual early-late promoter, the bacteriophage T7 RNA polymerase regulated by the lac operator and a vaccinia virus late promoter, and an HA epitope-tagged A28 under the control of the T7 promoter, which was also regulated by the lac operator (Fig. ). The lac repressor inhibited two consecutive steps, expression of T7 polymerase and of A28, ensuring high stringency of repression. The GUS gene was coinserted to allow selection of the recombinant virus, which was clonally purified. Controlled expression of A28 was achieved by addition of the desired concentration of IPTG. vA28-HAi formed plaques in the presence of IPTG (Fig. , lower left) but not in its absence (Fig. , upper left). At concentrations of 50 μM IPTG or higher, the plaques were similar in size to those of the parental virus, vT7lacOI (not shown). Although vA28-HAi did not form plaques in the absence of IPTG, at higher magnification we could detect single infected cells by their staining with antibody to vaccinia virus proteins (Fig. , upper right). Cell-to-cell spread occurred only in the presence of IPTG (Fig. , lower right). These data indicated that without IPTG, vA28-HAi exhibited a block in the formation or spread of infectious virus.
FIG. 6. Effect of IPTG on replication of vA28-HAi. (A) Diagram of relevant portions of the vA28-HAi genome. The locations of the A29L, A28L-HA, A27L, and GUS ORFs are shown. P11, a vaccinia virus late promoter; P7.5, a vaccinia virus early-late promoter; PA28L (more ...)
To differentiate between the above possibilities, we determined the yield of vA28-HAi in the presence or absence of IPTG under one-step growth conditions. The replication of vA28-HAi was entirely dependent on the addition of IPTG, as no increase in the amount of vA28-HAi was detected in the absence of inducer during a 24-h period (Fig. ). In contrast, in the presence of 100 μM IPTG, the amount of vA28-HAi increased >100-fold during this time. The kinetics of replication of vA28-HAi in the presence of IPTG was similar to that of the parental vT7lacOI (data not shown), except that the total accumulation of vA28-HAi was slightly reduced. The yield of vA28-HAi increased dramatically when the concentration of IPTG was raised from 0 to 50 μM; a further rise in the IPTG concentration to 1 mM had no effect on the virus yield (not shown). The dependence of A28 synthesis on IPTG is demonstrated elsewhere (16a
Late-protein synthesis and processing of the major core proteins occur normally in the absence of A28.
To determine whether virus replication was inhibited at an early or late stage, cells were infected with vA28-HAi in the presence or absence of inducer and were metabolically labeled with [35
S]methionine at various times. The proteins were then analyzed by SDS-PAGE and autoradiography. We could discern no difference in the patterns of viral-protein bands or in their intensities in the presence or absence of IPTG at any time examined (Fig. , top, and data not shown). Furthermore, the p4a, p4b, and p28 core protein precursors, derived from the A10R, A3R, and L4R ORFs, were processed into their mature products, 4a, 4b, and a 25-kDa protein, during the chase (Fig. , top). Processing was confirmed by Western blotting with specific antibody that recognizes p4b and 4b. Both the precursor and product were detected at 12 and 24 h in the presence or absence of inducer (Fig. , bottom). Since the processing of core proteins is dependent on morphogenesis (9
), these data suggested that morphogenesis occurred normally.
FIG. 7. Synthesis of viral proteins in cells infected with vA28L-HAi in the presence (+) or absence (−) of IPTG. BS-C-1 cell monolayers were infected with vA28L-HAi at a multiplicity of 5 PFU per cell in the presence or absence of 100 μM (more ...) Intracellular and extracellular virions are made in the absence of A28.
To directly examine the effect of A28 repression on morphogenesis, thin sections of cells infected with vA28-HAi in the presence or absence of IPTG were examined by transmission electron microscopy. Cells infected with vA28-HAi in the absence of IPTG showed the full range of viral structures, including IMV (Fig. ) and cell-associated enveloped virions (CEV) (Fig. ), which were indistinguishable from those produced by vA28-HAi in the presence of IPTG or wild-type virus (not shown). Our finding of mature virus particles was consistent with the observed normal processing of core proteins. Nevertheless, this result was unusual because the essential proteins of the IMV membrane that have been studied are needed for virus maturation.
FIG. 8. Electron microscopy of cells infected with vA28-HAi in the absence of IPTG. BS-C-1 cell monolayers were infected with vA28-HAi in the absence of IPTG for 24 h, fixed, and embedded in EPON, and ultrathin sections were prepared. (A) IMV in the cytoplasm. (more ...)
Our finding that the infection could not spread to neighboring cells in the absence of IPTG (Fig. ) seemed at odds with the detection of extracellular virus by electron microscopy (Fig. ). Because cell-to-cell spread is mediated primarily by virions at the tips of actin tails, we analyzed HeLa cells that were infected with vA28-HAi in the presence or absence of IPTG by confocal microscopy. After 24 h of incubation, the cells were fixed, and the still unpermeabilized cells were stained with a monoclonal antibody to the extracellular domain of the B5 protein component of CEV and extracellular enveloped virions. After being washed, the cells were permeabilized and stained to detect actin filaments and DNA. The B5 protein was detected as punctate green fluorescence on the surfaces of cells infected in the presence and absence of IPTG (Fig. ). Furthermore, numerous red actin tails with CEV at their tips were detected in both preparations. Thus, the failure of virus spread was not due to a defect in actin tail formation.
FIG. 9. Detection of CEV and actin tails by confocal microscopy. HeLa cells were infected with vA28-HAi in the presence (+) or absence (−) of IPTG. After 24 h, the cells were fixed and stained with anti-B5 monoclonal antibody, followed by fluorescein (more ...)