Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Helena Browne
hb100{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Present address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK.
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MAIN TEXT |
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Sequences encoding the TM and cytoplasmic tail regions of the human CD8 molecule, corresponding to the C-terminal 53 residues, were derived from the plasmid pS84 (a gift from S. Munro, LMB, Cambridge, UK) and were fused in-frame to sequences encoding the gD ectodomain (aa 1340) from HSV-1 strain Patton in the plasmid pCDNA3 (Invitrogen). The resulting construct was called pCDNAgDCD8CD8. The sequence encoding the gpi-anchor domain of decay-accelerating factor (DAF), which is composed of the C-terminal 37 aa residues, was derived from the plasmid gD1-DAF (Zurzolo et al., 1993) and was also fused in-frame with the sequence encoding the ectodomain of gD in a pCDNA3-based vector to generate pCDNA3gDDAF. Replacement of the authentic gD TM domain with that of either CD8 or DAF had no effect on the expression of these chimeric molecules on the plasma membrane of transfected cells (Fig. 1
a).
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However, a further possible interpretation of these findings is that pseudotyped virions containing gpi-linked gD enter cells more slowly because they incorporate less gD into their envelopes. To address this issue, we constructed a recombinant virus expressing gpi-linked gD so that we could determine whether purified virions contained wild-type amounts of the gDDAF molecule.
The open reading frame encoding gDDAF was isolated from plasmid pCDNA3gDDAF as a HindIIIXbaI fragment and ligated into the vector pINGHinc11gD (Whiteley et al., 1999). This contains HSV-1 sequences flanking the gD gene from nt 136 449 to 140 555, according to the numbering of nucleotides in the HSV-1 genome (McGeoch et al., 1988
); the resulting construct was called pINGHincgDgpi. BHK cells were co-transfected, using the method of Chen & Okayama (1987)
, with pINGHincgDgpi and with DNA extracted from cells infected with a gD-negative derivative of HSV-1 strain SC16, SCgDdelZ (Whiteley et al., 1999
). At 5 days after transfection, small plaques were observed and these were harvested and used to grow stocks of the recombinant virus, SCgDgpi. Stocks of this virus were assayed on a gD-helper cell line and extracellular virions were purified from infected BHK cells on Ficoll gradients, as described by Rodger et al. (2001)
. Numbers of virus particles were estimated by comparison with latex particles of known concentrations using negatively stained preparations, as described by Watson et al. (1963)
. The particle to infectivity ratio of SCgDgpi was estimated to be approximately 1000 : 1, whereas a preparation of purified wild-type SC16 virions had a ratio of 40 : 1.
The gD content of these virions was compared with that of wild-type virions by Western blotting serial twofold dilutions of equivalent numbers of virus particles using an anti-gD monoclonal antibody, LP14 (Minson et al., 1986). Parallel samples were immunoblotted with an antibody that recognizes the tegument protein VP16 as an internal control for loading equivalence; results are shown in Fig. 2
. It is notable that the electrophoretic characteristics of gpi-linked gD are slightly different from the wild-type molecule and it appeared to migrate as a more diffuse species. The amounts of gD and VP16 in both wild-type and SCgDgpi virions were, therefore, semi-quantified by densitometric analysis of the Western blot using a ChemiImager 4000 (Flowgen). These data, which are also presented in Fig. 2
, show that there are no significant differences between the amounts of gpi-linked gD and the amounts of wild-type gD that are incorporated into the virion envelope during assembly.
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This is not the first report of a gpi-anchored alphaherpesvirus gD molecule: Liang et al. (1995) produced a stably transfected cell line that expressed a gpi-anchored form of bovine herpes virus type 1 (BHV-1) gD. In agreement with our findings, they noted that gpi-anchored BHV-1 gD rescued the infectivity of a gD-negative virus. They also reported that a gD-null virus could enter cells expressing this hybrid molecule, implying that gDDAF, unlike wild-type gD, could act in trans to mediate the entry of virions lacking gD. However, these studies did not address the issue of whether gpi-anchored BHV-1 gD was less efficient than wild-type gD at mediating cellcell fusion nor whether virions containing this chimera could enter cells at rates similar to those with wild-type gD in their envelopes; whether this is a common property of alphaherpesvirus gD molecules is, at present, unclear.
A further possible implication of the observations described in this report concerns the mechanism by which HSV glycoproteins assemble into virions during morphogenesis. It is believed that the virus acquires its envelope glycoproteins at a post-endoplasmic reticulum compartment (either the trans-Golgi network or an early endosome) (Enquist et al., 1998; Harley et al., 2001
; Skepper et al., 2001
; Whiteley et al., 1999
), although the signals that direct incorporation of at least ten membrane proteins into the particle are not well understood. The results presented here show that gD assembles efficiently into virions in the absence of its authentic TM and cytoplasmic tail domains and that it is also incorporated when anchored in membranes by a gpi sequence. The gpi-anchor region of DAF has been shown to target the ectodomains of heterologous molecules to membrane microdomains, known as lipid rafts, which are enriched in cholesterol and sphingolipids (Friedrichson & Kurzchalia, 1998
). And, as a gDDAF hybrid molecule is incorporated into virus particles, it is possible that such membrane microdomains may represent a site of HSV-1 glycoprotein accumulation during budding, as has been shown to be the case for the envelope proteins of measles and influenza viruses (Manie et al., 2000
; Scheiffele et al., 1999
).
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ACKNOWLEDGEMENTS |
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Received 12 December 2002;
accepted 16 January 2003.