Two-hybrid analysis of the interaction between the UL52 and UL8 subunits of the herpes simplex virus type 1 helicase–primase

Nicoleta Constantin1 and Mark S. Dodson1

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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Herpes simplex virus type 1 expresses a heterotrimeric helicase–primase, the subunits of which are encoded by the viral UL5, UL8 and UL52 genes. The interactions of the UL52 protein with the UL8 and UL5 proteins were analysed by using the yeast two-hybrid system. The UL52–UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52–UL8 interaction gave a strong signal in the two-hybrid system. Deletion analysis showed that a 548 amino acid fragment of UL52 (amino acids 366–914) retains the ability to interact with UL8 and that the N-terminal 349 amino acids are dispensable for the interaction. A fragment library screen and co-immunoprecipitation experiments confirmed the deletion analysis results.


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Herpes simplex virus type 1 (HSV-1) encodes seven proteins that are necessary and sufficient for replication of viral DNA in host cells (Challberg, 1986 ; Challberg & Kelly, 1989 ). These proteins include a homodimeric origin-binding protein, a heterodimeric DNA polymerase, a single-stranded DNA-binding protein and a heterotrimeric DNA helicase–primase. The helicase–primase is composed of three subunits, encoded by the UL5, UL8 and UL52 genes (Crute & Lehman, 1991 ; Crute et al., 1989 ). UL5 is associated with the helicase activity and UL52 is associated with the primase activity (Dracheva et al., 1995 ; Gorbalenya & Koonin, 1993 ; Graves-Woodward et al., 1997 ; Graves-Woodward & Weller, 1996 ; Klinedinst & Challberg, 1994 ; Zhu & Weller, 1992 ). A heterodimeric subassembly consisting of the UL5 and UL52 proteins exhibits both helicase and primase activities (Calder & Stow, 1990 ; Dodson & Lehman, 1991 ). In the absence of UL52, purified UL5 is inactive (Calder & Stow, 1990 ; Sherman et al., 1992 ), indicating that the interaction between UL5 and UL52 is critical for the helicase activity of the complex. Purification of UL52 has so far not been reported, due to its insolubility when overexpressed alone; thus it has not been possible to determine whether UL52 can function as a primase in the absence of UL5 (Dodson & Lehman, 1991 ). The UL8 protein lacks any known catalytic or DNA-binding activity but is essential for virus replication (Carmichael & Weller, 1989 ; Parry et al., 1993 ). UL8 is required for transport of the UL5–UL52 subassembly into the nucleus and also interacts with other HSV-1 replication proteins within the replisome (Barnard et al., 1997 ; Calder et al., 1992 ; Falkenberg et al., 1997 ; McLean et al., 1994 ; Marsden et al., 1996 , 1997 ). Within the helicase–primase complex, UL8 stimulates both the DNA helicase and primase activities of the UL5–UL52 subassembly (Falkenberg et al., 1997 ; Sherman et al., 1992 ; Tenney et al., 1994 ). Thus, UL8 interactions with the UL5 and UL52 subunits of the helicase–primase seem to play an important role in modulating the helicase–primase activity and integrating this activity into the whole function of the replisome. Immunoprecipitation experiments indicate that a central 130 amino acid segment of UL8 may interact with both UL5 and UL52 (Barnard et al., 1997 ). Immunoprecipitation experiments also show that interactions occur among all three subunits of the heterotrimeric enzyme (McLean et al., 1994 ).

Specific interactions among UL5, UL8 and UL52 subunits are critical for the catalytic activity of the helicase–primase. Identification and characterization of the sites of these interactions are therefore crucial for understanding the overall mechanism of the HSV-1 helicase–primase. In addition, the sites of interaction may be suitable targets for the design of drugs that would function to disrupt protein–protein contacts required for the function of the helicase–primase.

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 helicase–primase. 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 EagI–BamHI fragment), pVL941/UL8 (as a SacI–BamHI 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 A5–B52) and between UL8 and UL52 (pair A8–B52). The UL52–UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52–UL8 interaction gave a strong signal in the two-hybrid system.


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Table 1. Fusion constructs used in the two-hybrid analysis

 


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Fig. 1. Two-hybrid analysis of the helicase–primase complex. (a) Identification of interactions among UL5, UL52 and UL8. A–B fusions were assayed pairwise for interaction by using both the colony-lift and liquid assays. In the colony-lift assay, interactions were scored as positive (+) (blue colonies) or negative (-) (white colonies). For the liquid assay, results are given as Miller units of ß-gal activity. Assays were performed in triplicate for each experiment. Interactions of the B52C deletion with A5 and A8 are also shown. (b) Deletion analysis of the UL52 gene. Several UL52 gene constructs containing deletions were fused to the GAL4 DNA-binding domain. All deletion fusions were assayed for interaction with A8 by using both the colony-lift and liquid assays. Results from the random fragment library screen are also shown (B52-lib #2).

 
In order to localize domains of UL52 that interact with UL8 or UL5, we constructed a series of deletions in the 1058 amino acid UL52 sequence (Table 1). We then assayed the UL52 deletion constructs fused to the GAL4 binding domain (B52 deletion fragments) for interaction with UL5 and UL8 fused to the GAL4 activation domain (A5 and A8). A C-terminal fragment of UL52 containing amino acids 350–1058 (B52C) retained the ability to interact with A8 (A8–B52C in Fig. 1a), suggesting that the C-terminal two-thirds of UL52 contains a region involved in the interaction with UL8. The same UL52 fragment (B52C) did not produce any signal in the two-hybrid system when assayed in the presence of A5 (A5–B52C in Fig. 1a), suggesting that the N-terminal 349 amino acids of UL52 may be required for the interaction with UL5. However, the A5–B52C result may be a false negative, since the A5–B52 interaction itself produced only a weak signal (0·5 Miller units in Fig. 1a) and other deletion constructs carrying the N terminus failed to produce a clear signal with A5 (results not shown). On the other hand, the negative result for the A5–B52C interaction could not be attributed to expression, stability or folding problems with the B52C construct, since this construct clearly interacted with A8 in the two-hybrid system. Either the N terminus of UL52 is required for the interaction with UL5 or the two-hybrid signal for this particular interaction is below the detection limit of our system.

Further deletion analysis allowed us to narrow down the UL52 interaction site with UL8 to within residues 350–914 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 542–720) 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. A–B52-lib #2 did not activate the reporters). This clone encoded a polypeptide corresponding to amino acids 366–1058 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 350–914). Toward this end, we expressed the UL52 fragment encoding amino acids 350–914 in a baculovirus system. A 1·7 kb EcoRI–EcoNI UL52 fragment (from B52CN3 in Table 1) was subcloned into the EcoRI/BglII sites of the baculovirus vector pBacPAK9 (Clontech) to make pBacPAK9/UL52{Delta}. A duplex oligonucleotide containing a start codon was cloned into the BamHI/EcoRI sites of pBacPAK9/UL52{Delta} in front of the UL52 sequence, and this plasmid was used to generate the recombinant baculovirus AcMNPV/UL52{Delta} as described previously (Summers & Smith, 1987 ). The UL52{Delta} protein was expressed and labelled in Sf9 cells infected with AcMNPV/UL52{Delta} 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{Delta} were first pre-cleared by immunoprecipitation with non-immune rabbit serum and Protein A–Sepharose 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 SDS–PAGE 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{Delta} from extracts expressing UL52{Delta} 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{Delta} co-precipitated with UL8 from extracts derived from cells that were doubly infected with baculoviruses recombinant for AcMNPV/UL8 and AcMNPV/UL52{Delta} (Fig. 2, lane 2). The control non-immune serum did not precipitate UL52{Delta}, UL52 or UL8 (Fig. 2, lanes 1 and 3). We conclude that UL52{Delta} contains a domain that interacts specifically with UL8.



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Fig. 2. Co-immunoprecipitation of a UL52 fragment (UL52{Delta}) with UL8. Labelled extracts from cells expressing UL52{Delta} and UL8 (52{Delta}+8), UL52 and UL8 (52+8), UL52{Delta} alone (52{Delta}), UL8 alone (8) or UL52 alone (52) were immunoprecipitated with anti-UL8 antibodies ({alpha}8) or non-immune serum ({alpha}-). The mobilities of UL8, full-length UL52 and UL52{Delta} (amino acids 350–914) are shown to the left.

 
The combined results of the two-hybrid analysis (deletion analysis and fragment library assay) and the immunoprecipitation experiments indicate that the UL52 region encompassing amino acids 366–914 is essential for the interaction with UL8, whereas the N-terminal 349 amino acids are not required for the interaction with UL8. The UL52 N terminus may participate in interactions with other replication proteins such as UL5. Additional experiments are needed to clarify the role of the UL52 N terminus. It is apparent from our results that the UL52 domain of interaction with UL8 does not consist of a small, contiguous sequence of amino acids, but it is rather contributed by amino acids from distant parts of the sequence (amino acids 366–914) that fold together. The two-hybrid analysis results provide a low-resolution map of the interaction between UL52 and UL8. Additional experiments are under way to characterize further the role of this interaction in the function of the helicase–primase complex.


   Acknowledgments
 
We thank Dr I. R. Lehman for UL8 antiserum and Dr Lauren Murata, Dr Jennifer Hall and Robert Baker for advice and discussion. This work was supported by grant RPG9705601NP from the American Cancer Society.


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Received 25 February 1999; accepted 9 June 1999.



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