Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA1
Author for correspondence: Duncan Wilson. Fax +1 718 430 8567. e-mail wilson{at}aecom.yu.edu
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Abstract |
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Introduction |
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One reagent that has been used to study HSV assembly is the drug brefeldin A (BFA). BFA is a fungal metabolite that has multiple effects upon the organelles of the secretory pathway, including inhibition of trafficking from endoplasmic reticulum (ER) to the Golgi apparatus, fusion of the cis, medial and trans cisternae of the Golgi with the ER (and subsequent disappearance of the Golgi) and fusion of the TGN with endosomes (Pelham, 1991 ; Sciaky et al., 1997
). In principle, BFA treatment might be expected to provide some information regarding the pathway of HSV egress. The single envelopment model predicts that enveloped virions should accumulate in the perinuclear space and ER, since their export from the ER via the classical secretory pathway would be blocked. Such a phenotype is clearly seen following short-term BFA treatment of pseudorabies virus (PRV)-infected cells (Whealy et al., 1991
); however, the extent to which these data can be used to distinguish between the various models for PRV egress has subsequently been questioned (Enquist et al., 1998
). In contrast, the re-envelopment model predicts that naked capsids should accumulate in the cytoplasm, because of the effect of BFA on the organelles (Golgi/TGN/endosomes) utilized for re-envelopment. In practice, both phenotypes have been reported for BFA-treated, HSV-infected cells (Koyama & Uchida, 1994
; Chatterjee & Sarkar, 1992
; Cheung et al., 1991
). One possible explanation for this complex result is that, in many of the earlier HSV studies, cells were exposed to BFA for extended periods of time (between 12 and 24 h). BFA is a cytotoxic drug (Jensen et al., 1995
; Ishii et al., 1989
; Yan et al., 1994
; Argade et al., 1998
) and, over such time-courses, could well have pleiotropic effects upon the biology of both the virus and the infected cell.
In earlier studies, we developed a model system to dissect late events in HSV assembly. The HSV-1 strain tsProt.A carries a reversible temperature-sensitive lesion in UL26, which encodes the maturational protease Pra (Rixon et al., 1988 ; Gao et al., 1994
; Preston et al., 1983
). At the non-permissive temperature of 39 °C, tsProt.A-infected cells accumulate immature nuclear procapsids (Rixon & McNab, 1999
; Newcomb et al., 2000
). Following downshift to the permissive temperature of 31 °C, these procapsids recruit the capsid subunit VP26 (Chi & Wilson, 2000
), package DNA (Preston et al., 1983
; Church et al., 1998
; Dasgupta & Wilson, 1999
) and give rise to exocytosing infectious particles in a synchronous wave (Church & Wilson, 1997
; Harley et al., 2001
). We have used this assay system to test the effect of various compounds upon HSV assembly by adding the drugs only at the time of temperature downshift (Church et al., 1998
; Dasgupta & Wilson, 1999
; Harley et al., 2001
). In this way, incubations can be for short periods of time, and any observed effect must be due to the action of the drug upon assembly and not upon other processes such as DNA synthesis or gene expression.
In the current study, we used this assay system to test the primary effect of BFA upon HSV assembly. Our results confirm earlier findings that BFA blocks p.f.u. production. However, in contrast to those studies, we found the assembly defect to be at the point of formation of perinuclear enveloped virus particles. Under our conditions, packaged capsids became trapped within the nucleoplasm and failed to bud into the perinuclear space and traffic into the cytoplasm. We conclude that the immediate effect of BFA upon the assembly pathway of HSV is to block capsid envelopment at the inner nuclear membrane.
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Methods |
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Scoring levels of infectious progeny virus and measurement of packaging and envelopment.
In order to measure the number of infectious progeny, cells were scraped up and sonicated and the resulting extracts were titrated on pre-formed Vero cell monolayers. Quantification of packaged capsids and enveloped packaged capsids by DNase I protection assay was done as described previously (Harley et al., 2001 ), except that radiolabelling of the viral DNA was achieved by incubation with 10 µCi/ml [3H]thymidine (New England Nuclear). Measurement of DNA packaging by Southern blot and probing with a 32P-radiolabelled SQ junction probe were done as described previously (Church et al., 1998
).
Preparation of nuclear extracts and post-nuclear supernatants.
Infected cells were washed twice with ice-cold homogenization buffer (250 mM sucrose, 10 mM TrisHCl, pH 7·6, 2 mM MgCl2), scraped up, pelleted and resuspended in the same buffer. Cells were gently broken by repeated passage through a 25 gauge needle and the nucleus (N) and post-nuclear supernatant (PNS) were separated by centrifugation at 2000 g for 10 min.
Electron microscopy.
Infected Vero cells were fixed and processed for electron microscopy as described previously (Church & Wilson, 1997 ).
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Results |
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Discussion |
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It has been demonstrated that, upon treatment with BFA, the Golgi apparatus fuses with the ER (Sciaky et al., 1997 ). Once this occurs, the nuclear membranes, which are contiguous with the ER, become flooded with Golgi proteins and lipids. We speculate that this dramatic alteration in the biochemical composition of the inner nuclear membrane could render it less suitable for budding of the HSV capsid, thereby resulting in accumulation of unenveloped nuclear capsids. To test this possibility, it will be necessary to examine the kinetics with which the lipid composition of the inner nuclear membrane changes during BFA treatment and to compare this with the time-point at which capsids begin to accumulate in the nucleoplasm. Unfortunately, such an experiment is impossible at present, since no techniques exist to isolate inner nuclear membrane lipids in the absence of the outer nuclear membrane and ER.
Why should short-term BFA treatment lead to accumulation of non-enveloped HSV in the nucleoplasm and of enveloped PRV in the perinuclear space and ER, as reported by Whealy et al. (1991)? One possible explanation is that the PRV study used asynchronous infections, wherein cells contain virions at every stage of assembly. Under these conditions, even if BFA addition blocked PRV capsid envelopment at the inner nuclear membrane, one would expect there already to be enveloped virions in the perinuclear space, formed prior to addition of the drug (which would be absent in our studies, where assembly is blocked at the procapsid stage). If, in addition to blocking envelopment at the inner membrane, BFA blocked de-envelopment at the outer membrane or export from the ER, these enveloped particles would be expected to accumulate, as observed by Whealy et al. (1991)
. Alternatively, whatever the reason for inhibition of HSV envelopment at the inner nuclear membrane, there is no reason to assume that the effect on PRV would be as severe. In this context, it is worth noting that, even for HSV, perinuclear virions do accumulate upon long-term BFA treatment, indicating that the envelopment process is only slowed, not blocked completely. There are also reasons to believe that HSV and PRV may differ in the details of their envelopment apparatus (Enquist et al., 1998
). The PRV gene UL3.5, for example, has been suggested to play an essential role in cytoplasmic envelopment (Fuchs et al., 1996
), but has no homologue in HSV. Similarly, mutations in UL20 cause HSV to accumulate in the perinuclear space (Baines et al., 1991
) but cause PRV to accumulate in cytoplasmic vesicles (Fuchs et al., 1997
).
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Acknowledgments |
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References |
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Received 2 January 2001;
accepted 7 March 2001.