Department of Biochemistry, University of Arizona, Biological Sciences West Building, 1041 E. Lowell Street, Tucson, AZ 85721-0088, USA1
Author for correspondence: Mark Dodson.Fax +1 520 621 9288. e-mail dodson{at}u.arizona.edu
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Abstract |
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Specific interactions among UL5, UL8 and UL52 subunits are critical for the catalytic activity of the helicaseprimase. Identification and characterization of the sites of these interactions are therefore crucial for understanding the overall mechanism of the HSV-1 helicaseprimase. In addition, the sites of interaction may be suitable targets for the design of drugs that would function to disrupt proteinprotein contacts required for the function of the helicaseprimase.
Our aim was to use the yeast two-hybrid system (Fields & Song, 1989 ) as a tool for mapping regions of contact that are made among the subunits of the HSV-1 helicaseprimase. We first determined which interactions could be detected with this system, using fusions between the HSV-1 proteins and the separated domains of the yeast GAL4 transcriptional activator. Interactions between the HSV-1 proteins reconstituted the yeast GAL4 transcriptional activator and resulted in expression of a lacZ reporter gene under the control of the yeast GAL4 elements. UL5, UL8 and UL52 DNA sequences were obtained from the plasmids pVL941/UL5 (as an EagIBamHI fragment), pVL941/UL8 (as a SacIBamHI fragment) and pVL941/UL52 (as a BamHI fragment), respectively (Dodson et al., 1989
), and cloned into yeast expression vectors pGBT9 and pGAD424 (Bartel et al., 1993a
). The resultant plasmids expressing the HSV-1 proteins as fusions with the GAL4 DNA-binding domain (B52) or the GAL4 activation domain (A5, A52, A8) (Table 1
) were co-transformed into the yeast strain SFY526 (Bartel et al., 1993b
), which carried the lacZ reporter gene. Co-transformants expressing pairs of A and B fusion proteins were first assayed for ß-galactosidase (ß-gal) activity by means of a colony-lift assay (Breeden & Nasmyth, 1985
). Colonies were lysed in the presence of X-Gal, and those that turned blue were scored as positive for an interaction (indicated by + in Fig. 1a
). The extent of ß-gal activity in each transformed pair was also quantified by a liquid assay (Chien et al., 1991
) using o-nitrophenyl ß-d-galactopyranoside as the substrate (numbers in Fig. 1a
). All assays were performed in triplicate in at least two independent experiments. We detected interactions between UL5 and UL52 (pair A5B52) and between UL8 and UL52 (pair A8B52). The UL52UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52UL8 interaction gave a strong signal in the two-hybrid system.
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Further deletion analysis allowed us to narrow down the UL52 interaction site with UL8 to within residues 350914 of UL52 (Fig. 1b). Two-hybrid interactions were lost when the N terminus was deleted beyond amino acid 350 (B52C2, B52C3) and when an internal segment (amino acids 542720) containing the catalytic site for primase activity was removed (B52-int1). Deletion of 78 amino acids from the UL52 C terminus (B52N4) resulted in loss of interaction with A8, initially suggesting that the extreme C terminus of UL52 might be required for interaction with UL8. However, the interaction with A8 was restored in double deletion mutants in which both the N-terminal 349 amino acids and the C-terminal 78 or 144 amino acids were removed from UL52 (B52CN4, B52CN3). Deletion of additional amino acids from the C terminus of UL52 (B52CN2) abolished the interaction with A8.
In order to localize further the UL52 domain that interacts with UL8, we constructed a DNA library of random UL52 gene fragments fused to the GAL4 DNA-binding domain (B52-lib). The UL52 gene was isolated from pVL941/UL52 as a 3·4 kb BamHI fragment and partially digested with restriction enzymes DpnI, BstUI and HaeIII, which cut at multiple sites within the fragment. The resulting blunt-ended fragments were cloned into the GAL4 DNA-binding domain vector pAS2-1 (Clontech), generating ligations in all three possible reading frames. The library and the plasmid encoding the A8 fusion were co-transformed into the yeast strain HF7c (Feilotter et al., 1994 ), which contains two reporter genes (HIS3 and lacZ) under the control of GAL4 elements. Transformants were grown on plates of synthetic medium lacking histidine. His+ colonies were isolated and lift-assayed for ß-gal activity. The His+/blue colonies contained interactive fusions that activated both the HIS3 and lacZ reporter genes. Plasmids were isolated from these colonies and assayed for ß-gal activity in the yeast strain SFY526. Clone B52-lib #2 yielded a strong and specific signal with A8 and also lacked background activity (i.e. AB52-lib #2 did not activate the reporters). This clone encoded a polypeptide corresponding to amino acids 3661058 of UL52 (Fig. 1b
). Thus, the fragment library assay results were in agreement with the deletion analysis results and also enabled us to reposition the N-terminal boundary of the UL52 domain of interaction with UL8 from amino acid 350 to amino acid 366.
Immunoprecipitation was used to verify that UL8 interacts with the UL52 fragment contained in B52CN3 (amino acids 350914). Toward this end, we expressed the UL52 fragment encoding amino acids 350914 in a baculovirus system. A 1·7 kb EcoRIEcoNI UL52 fragment (from B52CN3 in Table 1) was subcloned into the EcoRI/BglII sites of the baculovirus vector pBacPAK9 (Clontech) to make pBacPAK9/UL52
. A duplex oligonucleotide containing a start codon was cloned into the BamHI/EcoRI sites of pBacPAK9/UL52
in front of the UL52 sequence, and this plasmid was used to generate the recombinant baculovirus AcMNPV/UL52
as described previously (Summers & Smith, 1987
). The UL52
protein was expressed and labelled in Sf9 cells infected with AcMNPV/UL52
in the presence of [35S]methionine (ICN Pharmaceuticals). Crude extracts were prepared as described previously (McLean et al., 1994
). Labelled extracts from cells infected with AcMNPV/UL52, AcMNPV/UL8 (Dodson et al., 1989
) and AcMNPV/UL52
were first pre-cleared by immunoprecipitation with non-immune rabbit serum and Protein ASepharose beads (Sigma). The supernatants were then immunoprecipitated with either polyclonal antibody against UL8 or additional non-immune serum (McLean et al., 1994
) and analysed by SDSPAGE and autoradiography.
The UL8 antiserum specifically precipitated UL8 from an extract derived from AcMNPV/UL8-infected Sf9 cells (Fig. 2, lane 6), but did not precipitate UL52
from extracts expressing UL52
alone (Fig. 2
, lane 5) or UL52 from extracts expressing UL52 alone (Fig. 2
, lane 7). A polypeptide exhibiting the mobility of full-length UL52 co-precipitated with UL8 when extracts were derived from Sf9 cells that were doubly infected with AcMNPV/UL8 and AcMNPV/UL52 (Fig. 2
, lane 4), as shown previously by McLean et al. (1994)
. A polypeptide exhibiting the predicted mobility of UL52
co-precipitated with UL8 from extracts derived from cells that were doubly infected with baculoviruses recombinant for AcMNPV/UL8 and AcMNPV/UL52
(Fig. 2
, lane 2). The control non-immune serum did not precipitate UL52
, UL52 or UL8 (Fig. 2
, lanes 1 and 3). We conclude that UL52
contains a domain that interacts specifically with UL8.
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Acknowledgments |
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References |
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Received 25 February 1999;
accepted 9 June 1999.
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