Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Author for correspondence: Geoffrey Smith. Present address: WrightFleming Institute, Imperial College School of Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK. Fax +44 20 7594 3973. e-mail glsmith{at}ic.ac.uk
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Abstract |
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Introduction |
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VV forms clear round plaques in many different cell lines and some strains of VV form comet-shaped plaques under liquid overlay (Appleyard et al., 1971 ; Payne, 1980
), like herpes simplex virus (HSV) (Shinkai, 1975
). This characteristic plaque phenotype is caused by the efficient long-range spread of virus, resulting in a series of secondary plaques (comet tails) distant from the primary infection site (comet heads). The comet-shaped plaque phenotype of VV can be blocked by antibodies (Ab) directed against EEV but not IMV (anti-comet assay) (Appleyard et al., 1971
; Appleyard & Andrews, 1974
; Payne, 1980
; Vanderplasschen et al., 1997
; Galmiche et al., 1999
). Anti-EEV, but not anti-IMV, Ab provides passive immunity against VV challenge (Madeley, 1968
; Appleyard et al., 1971
; Boulter et al., 1971
; Turner & Squires, 1971
; Appleyard & Andrews, 1974
; Galmiche et al., 1999
).
IMV is assumed to be the virion responsible for the spread of VV between hosts because the EEV membrane is too fragile to survive the physical environment outside the host, and once broken, will release a fully infectious and relatively stable IMV particle (Ichihashi, 1996 ). IMV and EEV possess a different set of virus proteins on their surfaces (Payne, 1978
, 1992
) and use different cellular receptors (Vanderplasschen & Smith, 1997
; Krijnse-Locker et al., 2000
) and pathways to enter cells (Payne & Norrby, 1978
; Ichihashi & Oie, 1980
; Vanderplasschen et al., 1998a
; Krijnse-Locker et al., 2000
).
CEV is physically indistinguishable from EEV and may be released from the cell surface by mild trypsin treatment (Blasco & Moss, 1992 ). The retention of CEV on the cell surface rather than its release as EEV contrasts with other viruses, e.g. measles virus, human immunodeficiency virus type 1 and influenza virus, where the cellular receptors are usually down-regulated or removed to facilitate virus release and prevent virus aggregation (Palese et al., 1974
; Firsching et al., 1999
; Piguet et al., 1999
). CEV mediates cell-to-cell spread, but the absolute level of CEV is not critical for plaque size because VV strains International Health Department (IHD)-J and Western Reserve (WR) form plaques of similar size despite WR retaining more CEV than IHD-J on the cell surface (Sanderson et al., 1998a
).
Several VV proteins are associated with only IEV (A36R and F12L) or with IEV/CEV/EEV (A33R, A34R, A56R, B5R and F13L). The study of virus mutants with these genes deleted or repressed, showed that these proteins are not needed for IMV production but are involved in the various stages of virus egress, such as wrapping of IMV, transport of IEV to the cell surface, actin tail formation, EEV release and plaque phenotype. The properties of these deletion mutants are summarized in Table 1. IEV proteins A36R (Röttger et al., 1999
; van Eijl et al., 2000
) and F12L (Zhang et al., 2000
; van Eijl et al., 2002
) are each non-glycosylated proteins that have the majority of their amino acids in the cytosol, whereas EEV proteins A33R (Roper et al., 1996
), A34R (Duncan & Smith, 1992
), A56R (Shida & Dales, 1981
; Shida, 1986
) and B5R (Engelstad et al., 1992
; Isaacs et al., 1992
) are glycosylated integral membrane proteins with a type I (B5R and A56R) or type II (A33R and A34R) membrane topology. F13L is an acylated membrane-associated protein that is associated with the inner surface of the EEV outer envelope (Hiller & Weber, 1985
; Hirt et al., 1986
).
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Here, we have investigated the roles of IMV, IEV, CEV and EEV in VV spread in vitro, by using a panel of virus mutants lacking individual IEV- or EEV-specific genes, and Abs that neutralize IMV or EEV. We demonstrate that comet-shaped plaques are probably made by convection currents, that VV spreads from cell to cell by Ab-sensitive and Ab-resistant pathways, and that the A33R protein has a role in Ab-resistant spread.
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Methods |
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Antibodies.
Mouse monoclonal Ab (mAb) 2D5 against the IMV L1R protein (Ichihashi & Oie, 1996 ), mAb AB1.1 against the IMV D8L protein (Parkinson & Smith, 1994
), rabbit antiserum against the B5R protein (
-B5R) (Galmiche et al., 1999
) and VV-immune rabbit antiserum Rb-WR2 (Law & Smith, 2001
) were described previously. Antisera were heat-inactivated at 56 °C for 30 min before use.
Plaque assays.
(i) Liquid overlay for comet formation. IMV was diluted in DMEM/2% and adsorbed onto cells for 2 h at 37 °C. Unbound virus was washed away with PBS and the cells were overlaid with liquid medium (DMEM/2%) and stained 2 days later (unless specified otherwise) with 0·05% crystal violet in 15% ethanol. Antibodies and 10 µg/ml IMCBH (N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine) (Payne & Kristenson, 1979 ) were included in the overlays where indicated. (ii) Semi-solid overlay. As for (i) except that virus was adsorbed for 1 h and cells were overlaid with DMEM/2% containing 1·5% carboxymethylcellulose (CMC).
Titration of EEV.
The infectivity of EEV was quantified as described (Law & Smith, 2001 ). Briefly, fresh virus supernatants were collected at the indicated times, diluted and incubated with mAb 2D5 (diluted 1/2000) for 1 h at 37 °C to neutralize contaminating IMV. When specified, Rb-WR2 Ab was included to neutralize EEV. The virus was adsorbed onto cells for 1 h, washed, and overlaid with 1·5% CMC in DMEM/2%. After incubation, the plaques were stained as above.
Microscopy.
Methods for indirect immunofluorescent staining have been described elsewhere recently (Law & Smith, 2001 ). VV-infected cells were detected using mAb AB1.1 (5 µg/ml) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (diluted 1/100, Jackson Laboratories). The methods for electron microscopy have been described elsewhere recently (Hollinshead et al., 2001
).
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Results |
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VV cell-to-cell spread
Ab Rb-WR2 not only inhibited the long-range spread of all the mutants but also caused significant reduction in the plaque size of some of the mutants. These observations suggest there are at least two mechanisms of virus cell-to-cell spread that are either sensitive or resistant to Ab. This was studied further by comparing the plaque size in the presence of Ab against different forms of VV and under a semi-solid overlay so as to measure virus cell-to-cell spread only. Fig. 2(a) shows the plaques formed by the mutants 10 days p.i. in the presence of mAb 2D5 (diluted 1/500), Rb-WR2 or
-B5R antisera (diluted 1/100), and their sizes are quantified in Fig. 2(bf
). MAb 2D5 is an IMV-neutralizing Ab (Ichihashi, 1996
), Rb-WR2 inhibits both IMV and EEV (Law & Smith, 2001
) whereas
-B5R neutralizes only EEV (Galmiche et al., 1999
; Law & Smith, 2001
).
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Addition of mAb 2D5 had little effect on the plaque size of the mutants, although vF13L was reduced slightly more than the others (Fig. 2d
). Possibly, IMV was released late during infection with v
F13L and therefore its plaques were affected by mAb 2D5. These data indicate that IMV is not sufficient for VV cell-to-cell spread.
In contrast to mAb 2D5, the antiserum Rb-WR2 raised against a live infection had varying effects on the mutants (Fig. 2e). Most strikingly, plaques were not made by v
A33R in the presence of Rb-WR2. Rb-WR2 also reduced the plaque size of WR, v
A34R, v
A56R and v
B5R, but not v
A36R or v
F12L. Very faint plaques were seen with v
F13L, but these were difficult to quantify. These results allow the viruses to be divided into three groups: complete blockage of virus spread (v
A33R), partial blockage (WR, v
A34R, v
A56R, v
B5R and v
F13L) and no blockage (v
A36R and v
F12L). Since anti-IMV Ab did not affect cell-to-cell spread, the inhibition should be mediated by the anti-EEV Ab present in Rb-WR2. The blockage of plaque formation by v
A33R using Rb-WR2 is not due to an increase in Ab sensitivity of the mutant because EEV made by v
A33R showed similar sensitivity to neutralization by antibody (Law & Smith, 2001
) and further analyses using a range of dilutions of Ab confirmed that the sensitivity was the same as wild-type (Fig. 3
). On the other hand, EEV of v
B5R is less sensitive to Rb-WR2 because of the absence of major virus-neutralizing epitopes on the B5R protein (Law & Smith, 2001
). Interestingly, antibody enhancement was observed in both v
A33R and WR at similar Ab dilutions.
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Does abortive infection by vA33R occur in the presence of Rb-WR2?
The complete blockage of plaque formation by vA33R using Rb-WR2 Ab was unexpected. To investigate whether this blockage was a result of an Ab-sensitive spreading mechanism or a post-binding neutralization, we compared v
A33R- and v
A34R-infected cells by immunofluorescent microscopy (Fig. 4
). Without Ab (Fig. 4a
, d
) or with mAb 2D5 (Fig. 4b
, e
), extensive cytopathic effect, such as cell rounding, cell flattening and projection formation (Sanderson et al., 1998b
), was found in cells infected by either mutant. At 72 h p.i., the infected foci were the same size with or without mAb 2D5 and were about 0·24 mm and 0·38 mm for v
A33R and v
A34R, respectively. In the presence of Rb-WR2, cells infected with v
A34R developed similar cytopathic effect, although virus spread was restricted by the Ab (Fig. 4f
). With v
A33R in the presence of Rb-WR2 (Fig. 4c
), very few infected cells were found in all the infected foci examined. In addition, the infected cells did not develop extensive cytopathic effect and were not clustered together. The individual infected cells within these atypical foci were unlikely to have derived from several separate infections because only a few plaque-forming units were used to inoculate the cell monolayer. Possibly, virus-induced cell movement might have caused the infected cells to disconnect from each other after the spread of the virus.
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Is IMV wrapping important for the Ab-resistant spreading mechanism?
vA33R and v
A34R make higher levels of EEV (Table 1
) despite the wrapping of IMV being either incomplete (Roper et al., 1998
) or inefficient (Duncan & Smith, 1992
; Wolffe et al., 1997
). Therefore, we investigated whether an alternative mechanism for CEV and EEV formation, such as budding of IMV from the cell surface, occurred with v
A33R and resulted in the increased susceptibility to Rb-WR2.
The wrapping of IMV to form IEV can be inhibited pharmacologically by IMCBH (Kato et al., 1969 ; Payne & Kristenson, 1979
; Hiller et al., 1981
), which targets the F13L protein (Hiller et al., 1981
; Schmutz et al., 1991
). Fig. 5(a)
shows the plaques formed in the presence of IMCBH at 5 days p.i. As expected, IMCBH had no effect on v
F13L due to the absence of the target protein, while WR and v
A56R produced plaques similar in size to those of v
F13L. All other mutants were inhibited severely and only tiny infected foci were seen.
|
In the presence of IMCBH (Fig. 5b, black bars), the levels of EEV made by v
A33R and v
A34R were reduced drastically, suggesting EEV were formed by the normal IMCBH-sensitive pathway. Similar results were noted for v
B5R and v
F12L. Some EEV were still made by WR, v
A56R and v
F13L, consistent with the observations on virus cell-to-cell spread above. Surprisingly, a significant level of EEV was made by v
A36R in the presence of IMCBH, despite plaque formation being inhibited by this drug (Fig. 5a
). These data may suggest that the inhibition of EEV production by IMCBH is also partially dependent on the presence of A36R in addition to F13L.
The formation of IEV by vA33R and v
A34R was investigated further by electron microscopy. In RK13 cells infected with VV strain IHD-J most IMV wrapping occurred before 20 h p.i. (Payne & Kristenson, 1979
). Since v
A33R and v
A34R make enhanced levels of EEV at 1824 h p.i. (Table 1
), the deletion of A33R or A34R might have accelerated the kinetics for IMV wrapping and EEV release. Therefore, RK13 cells infected with v
A33R, v
A34R, WR and vRevA34R (the revertant virus of v
A34R) were analysed at 6 h and 8 h p.i. (Fig. 6
) and intracellular virions (IMV and IEV) were quantified (Table 2
). Fully wrapped IEV particles were found in cells infected with both mutants (Fig. 6
) but, compared to the other viruses, lower levels of IEV were found in v
A34R-infected cells at both 6 and 8 h p.i. This suggested that either IEV formation is less efficient or IEV dissemination is quicker. In all samples, we found no evidence that EEV were generated by budding and conclude that wrapping of IMV is necessary for the formation of CEV/EEV by v
A33R and v
A34R. Therefore, the sensitivity to Ab of v
A33R cell-to-cell spread is unlikely to be caused by an alternative pathway of CEV/EEV formation.
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Discussion |
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Long-range virus dissemination was analysed in cell culture by the formation of comet-shaped plaques and was shown to be mediated by EEV that is probably dispersed by convection currents to infect distant cells. By tilting the culture dish 10° all comets became parallel and went uphill. In vivo, EEV is released early in infection and is important for systemic spread (Payne, 1980 ).
The mechanism of cell-to-cell spread was investigated by a direct comparison of the plaques formed by all mutants lacking individual IEV or EEV proteins and the effects of anti-IMV and -EEV Ab. The plaque sizes of all mutants, except vA56R, was reduced at least threefold in diameter compared to WR. v
A56R formed syncytia (Ichihashi & Dales, 1971
; Sanderson et al., 1998a
) but had similar sensitivity to Ab compared with WR, indicating that syncytium formation did not contribute to virus spread. Syncytium formation by v
A56R occurs late during infection and might occur between infected cells rather than between infected and uninfected cells.
After vA56R, the next largest plaque is made by v
A36R followed by v
B5R, v
A34R, v
A33R, v
12L and v
F13L. All mutants make near normal levels of IMV (Table 1
), demonstrating that IMV is insufficient for plaque formation. Plaque size is also not determined by whether or not IMV are wrapped to form IEV because mutants that can (v
A36R) or cannot (v
F13L) make IEV each form a small plaque. The levels of CEV and EEV also are not important for plaque size since viruses IHD-J and WR produce very different levels of CEV but make similar plaque sizes, and viruses with either enhanced (v
A34R) or reduced (v
B5R) EEV levels each make small plaques (Table 1
). The major factor determining plaque size is the ability to make actin tails that mediate efficient cell-to-cell spread (Table 1
).
The mechanism(s) of VV spread were dissected further using neutralizing Ab specific to IMV or EEV. A reduction in plaque size suggests that the virus spreads from cell to cell in a pathway that is exposed to Ab, whereas, if the plaque size is unchanged the virus spreads in a pathway that is protected from Ab. Plaques made by all viruses were largely unaffected by anti-IMV Ab (Fig. 2d). Therefore, cell-to-cell spread must involve the enveloped virions CEV/EEV, and consistent with this, anti-EEV Ab had varying effects on the different mutants (Fig. 2e
, f
). In the presence of Rb-WR2 or
-B5R Ab, WR and v
A56R plaque sizes were inhibited to a similar extent (Fig. 2e
), but the plaques were still bigger than those made by other mutants, even in the absence of the Ab (Fig. 2b
). This reiterated the importance of actin tails for efficient cell-to-cell spread and showed that this mechanism of spread is resistant to Ab. Resistance to Ab was also observed in other mutants unable to make actin tails, e.g. v
A36R and v
F12L, and therefore an actin tail-independent and Ab-resistant pathway exists. Both A36R and F12L proteins are IEV-specific and not exposed to Ab, and so these results are unlikely to be due to the removal of neutralizing targets on CEV/EEV. Rb-WR2 reduced the size of plaques formed by v
A34R and v
B5R and only very faint plaques were formed by v
F13L, implying that these viruses used both Ab-sensitive and Ab-resistant pathways for their spread. Most dramatic was the abrogation of v
A33R plaque formation by Rb-WR2, showing this mutant spreads entirely via an Ab-sensitive pathway.
Anti-B5R Ab affected vA33R the most among all the mutants, showing B5R is a target in the Ab-sensitive pathway. However, other virus antigen(s) must be involved because
-B5R did not block v
A33R completely, affected v
A34R only marginally and had no effect on v
F13L (Fig. 2f
). IMV wrapping by v
F13L is inhibited severely and therefore this mutant makes very low levels of CEV and EEV (Table 1
). v
F13L was also slightly more sensitive to mAb 2D5 than other mutants (Fig. 2c
), suggesting that IMV might contribute to some levels of virus spread in this mutant. This might account for the resistance to
-B5R. Cell-to-cell spread of v
A34R was fairly resistant to
-B5R despite v
A34R making 19- to 25-fold higher levels of EEV. This suggested A34R is required for the Ab inhibition via B5R. A34R may have a role in the conformation, recruitment or function of the B5R protein.
In summary, these data show that VV uses a combination of mechanisms to spread between cells. The mechanisms can be divided into (i) actin tail-dependent Ab-resistant pathway (WR and vA56R); (ii) actin tail-independent Ab-resistant pathway (v
A36R and v
F12L); and (iii) Ab-sensitive pathway (v
A33R). Actin tails have a major role in the Ab-resistant pathway. This is probably because actin tails push the virions into the neighbouring cells directly without exposing the virions to Ab. WR and v
A56R spread by a combination of these three pathways whereas v
A34R, v
B5R and v
F13L use pathways (ii) and (iii).
The Ab-susceptibility of vA33R is analogous to HSV-1 gE and gI deletion mutants. HSV-1 entry kinetics and replication were not affected by deletion of either gene; however, cell-to-cell spread of these mutants was impaired (Dingwell et al., 1994
), and neutralizing Ab reduced the yields of the mutants but not wild-type virus. HSV gE and gI form a complex (Johnson et al., 1988
) that accumulates at sites of cellcell contact, possibly by interacting with junctional components (Dingwell & Johnson, 1998
), and which may mediate HSV transfer across the cell junctions. Similarly, varicellar-zoster virus gE expression in polarized epithelial cells altered the F-actin organization and accelerated the formation of tight junctions between cells (Mo et al., 2000
). The VV A33R protein expressed by Semliki forest virus or VV accumulated on microvillus-like cell surface projections (Lorenzo et al., 2000
). A33R might aid virus spread through cell junctions by interacting with junctional proteins in a similar fashion to HSV gEgI. A33R might also interact with surface molecules of neighbouring cells to facilitate cellcell contacts for virus spread. Deletion of A33R could disrupt these cellcell interactions and permit v
A33R to spread only in an Ab-sensitive pathway. Consistent with this proposal, the passive transfer of an anti-A33R mAb, and immunization with A33R DNA (Hooper et al., 2000
) or recombinant A33R protein (Galmiche et al., 1999
), protected mice from VV challenge. However, neither the anti-A33R mAb nor Ab raised against recombinant A33R proteins neutralized EEV in vitro.
The production of several infectious forms of VV is explained by these virions having different roles in the virus life-cycle. IMV is highly immunogenic (see JGV Online for supplementary data, http://vir.sgmjournals.org) (Law & Smith, 2001 ) and susceptible to neutralization by complement (Vanderplasschen et al., 1998b
), and therefore is poorly suited to virus spread within a host. It would be advantageous for the majority of VV infectivity (IMV) to be retained and protected within cells from Ab and complement. However, IMV particles are physically robust and are well suited for dissemination between hosts. To aid virus dissemination within a host, VV has exploited several features of the cell biology. It uses cellular membranes to wrap IMV particles and by the acquisition of host complement factors protects EEV particles from destruction by complement (Vanderplasschen et al., 1998b
), and uses virus-encoded proteins in the EEV outer envelope to bind to different receptors from IMV (Vanderplasschen & Smith, 1997
) and to enhance the range of cell types that may be infected.
The role of IEV is less characterized and might appear unnecessary, for to make CEV or EEV an IMV might bud through the plasma membrane. Indeed, limited budding occurs late during infection in VV strain IHD-W (Tsutsui, 1983 ) and in fowlpox virus (Boulanger et al., 2000
). The apparent inefficiency of IMV wrapping but enhanced EEV production by mutants v
A33R and v
A34R (Duncan & Smith, 1992
; McIntosh & Smith, 1996
; Roper et al., 1998
) further questioned the significance of IEV in the VV life-cycle. However, by pharmacological and microscopic approaches, we demonstrated here that wrapping not only occurs with these mutants but is an essential step for both mutants (Figs 5
and 6
). Two functions for IEV are proposed. First, asymmetric distribution of the A36R protein enables the unidirectional polymerization of actin after the outmost IEV membrane has fused with the plasma membrane (van Eijl et al., 2000
). This could not easily occur via budding. Second, both IMV and IEV utilize microtubules for intracellular movement (Sanderson et al., 2000
; Hollinshead et al., 2001
; Ward & Moss, 2001
). The wrapping of IMV by additional membranes places different proteins on the IMV and IEV surface, and consequently these virions may have different interactions with microtubule components enabling movement towards (IMV) or away from (IEV) the site of wrapping.
Finally, we propose an explanation for the existence of CEV. CEV is structurally indistinguishable from EEV, but with most VV strains many enveloped virions are retained on the cell surface rather than being released. This appears curious since many viruses enhance virus release by down-regulating cell receptors. However, the efficient cell-to-cell dissemination of virus requires actin tail formation from the cell surface, and so enveloped virions need to be retained long enough at the surface to promote this activity. A34R and B5R are involved in the retention of CEV on the cell surface. The deletion of the entire B5R gene or fusion with the VV A56R extracellular domain reduced wrapping and EEV release (Engelstad & Smith, 1993 ; Wolffe et al., 1993
; Mathew et al., 2001
) while deletion of any of the short consensus repeat domains of B5R enabled wrapping and increased EEV release (Herrera et al., 1998
; Mathew et al., 1998
). The deletion or mutation of A34R enhanced the release of EEV (Blasco et al., 1993
; McIntosh & Smith, 1996
) and reduced wrapping compared to WR and vRevA34R (Table 2
). B5R interacts with A34R (Röttger et al., 1999
) and, interestingly,
-B5R Ab did not inhibit the cell-to-cell spread of v
A34R (Fig. 2f
). A34R and B5R may work in concert to affect EEV release.
In summary, VV benefits from having four different virus forms, IMV, IEV, CEV and EEV, for efficient cell-to-cell spread (CEV and actin tails) and long-range spread (EEV) within the host, and reserving the majority of infectivity (IMV) for transmission between hosts.
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Acknowledgments |
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References |
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Received 29 July 2001;
accepted 20 September 2001.