The UL3 open reading frame of herpes simplex virus type 1 codes for a phosphoprotein

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Virus Research 44 (1996) 137 142

Virus Research

Short communication

The UL3 open reading frame of herpes simplex virus type 1 codes for a phosphoprotein Homayon Ghiasi a'b'*, Guey-Chuen Perng a, Steve Cai a, Anthony B. Nesburn a'b, Steven L. Wechsler a'b aOphthalmology Research--Davis 5069, Cedars-Sinai Medical Center Research Institute, D5069, 8700 Beverly Blvd., Los Angeles, CA 90048, USA bDepartment of Ophthalmology, UCLA School of Medicine, Los Angeles, CA 90024, USA

Received 29 January 1996; revised 4 April 1996; accepted 8 April 1996

Abstract

Based on sequence analysis, the protein encoded by the UL3 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encoding the complete sequence of the UL3 ORF in a baculovirus expression system. Western blotting was done using polyclonal antibody raised against synthetic UL3 peptides. Two major baculovirus-UL3 expressed protein bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 kDa were detected. None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated. Cell fractionation studies indicated that the UL3 protein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylation. In contrast, the baculovirus expressed UL3 protein was phosphorylated as judged by 32pi-labeling. Immunoprecipitation followed by S D S - P A G E demonstrated a single 32pi-labeled UL3 related band with an apparent molecular weight of 33 kDa, indicating that the UL3 protein was a phosphoprotein. Antibodies produced in mice vaccinated with baculovirus-UL3 protein reacted with two UL3 related HSV-1 bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3. Keywords: Herpes simplex virus type 1 (HSV-1); UL3; Phosphoprotein

* Corresponding author. Tel: + 1 310 8556455; fax: + 1 310 6528411.

The herpes simplex virus type 1 g e n o m e (HSV1) is a 152-kilobase (kb) linear duplex D N A

0168-1702/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII SO168-1702(96)01330-5

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molecule [20,25,27]. HSV-1 contains more than 75 genes that are expressed during productive infection. These genes are divided into at least three classes depending on their temporally regulated expression [11,20]. Included among these genes are 10 genes encoding HSV-1 glycoproteins [4,12,13,20,28]. Several additional HSV-1 open reading frames (ORFs) may code for as yet undescribed membrane proteins based on their sequence analysis [20]. A previously uncharacterized ORF, UL3, is the focus of this study. Based on DNA sequence analysis, UL3 appears to encode a protein of 235 amino acids (aa) with a predicted molecular weight of 25 607 Da [20]. The prediction of a potential N-linked glycosylation site, a potential signal sequence, and a potential hydrophobic transmembrane domain suggest that UL3 might be a glycoprotein [19,20]. Like several previously characterized glycoproteins, UL3 does not appear to be essential for HSV-1 replication in tissue culture [3]. Although the product of the HSV-1 UL3 gene has not yet been identified, while the work reported here was in progress, the HSV-2 UL3 protein was shown to be a nuclear localizing phosphoprotein [30]. To characterize the HSV-1 U L 3 0 R F , a recombinant baculovirus (vAc-UL3) was constructed that expresses the UL3 gene of HSV-1 by digestion of plasmid pSG1 [10] with EcoRI/XbaI. The 1957 bp fragment containing the UL3 gene was treated with Bal 31 and after addition of BamHI linkers, an 875 bp DNA fragment containing the UL3 gene was ligated into the unique Bam HI site of the vector pAcYM1 [18] to produce the transfer vector pAc-UL3. The orientation of UL3 was confirmed by restriction enzyme analysis. Partial sequence analysis showed that the pAc-UL3 clone has only five noncoding HSV-1 nucleotides before the first ATG of the open reading frame and 155 noncoding HSV-1 nucleotides after the UL3 termination codon at the 3' end. To transfer the UL3 gene into the baculovirus AcNPV genome, Sf9 cells were co-transfected with purified linearized baculovirus (AcNPV) DNA and pAc-UL3 plasmid DNA as previously described [15]. After two cycles of plaque purification, three polyhedrinnegative recombinants with similar properties were isolated. One of the recombinant viruses was

arbitrarily chosen for further study and designated vAc-UL3 (also referred to as baculovirusUL3). To verify the presence of full length HSV-1 UL3 DNA in vAc-UL3, the baculovirus-UL3 DNA was digested with the restriction enzyme BamHI and Southern blots were done using the UL3 gene as a probe (not shown). We report here the expression and preliminary characterization of the HSV-1 UL3 protein and show that it is a phosphoprotein, not a glycoprotein. To analyze the size of the baculovirus expressed UL3, confluent monolayers of St9 cells were infected at a multiplicity of 10 pfu/cell with the recombinant baculovirus-UL3 for 48 h. Total protein extracts were run on 12% SDS-PAGE and analyzed by Western blotting using rabbit polyclonal antisera against UL3 peptides. Synthetic peptides were made based on the published sequence of HSV-1 strain 17 [20]. Each peptide was 15 aa long and had a purity of more than 70%. An equal molar mixture of four peptides corresponding to aa 163-177 (KSLQMFVLCKRAHAA), 178-192 (RVREQLRVVIQSRKP), 193-207 (RKYYTRSSDGRLCPA), and 208222 (VPVFVHEFVSSEPMR) were coupled to bovine serum albumin (BSA) as described by the manufacturer (Pierce Chemical). Rabbits were injected intraperitoneally at five different sites with a total of 500 ¢tg of BSA-coupled peptide mixture in Freund's complete adjuvant on day 0. Additional sets of injections of 500 ¢tg of BSA-coupled peptide mixture in Freund's incomplete adjuvant were given on days 21, 42, and 63. Serum was collected 21 days after the fourth vaccination. The anti-UL3-peptide polyclonal antibody reacted with two major bands of 30 kDa and 31 kDa from baculovirus-UL3 infected cells (Fig. I(A), UL3 lanes; arrows). The anti-UL3-peptide polyclonal antibody also reacted with three additional minor UL3 specific bands of 29, 32, and 33 kDa. The anti-UL3 antiserum did not detect any of these bands in St9 cells (lane Sf9) or wild-type baculovirus infected Sf9 cells (lane Wt). To determine whether the expressed UL3 underwent glycosylation, infected cells were treated with 4 / l g tunicamycin/ml of media from 0-48 h post infection, and total cell extracts were analyzed by Western blots using anti-UL3-peptide

H. Ghiasi et al. / Virus Research 44 (1996) 137-142

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Fig. 1. (a) Western blot analysis of baculovirus expressed UL3. Sf9 cells were infected with the baculovirus-UL3 recombinant (baculovirus-UL3) at a multiplicity of infection of 10 pfu/cell for 48 h. Some infected cells were treated with tunicamycin throughout the infection. The infected cells were lysed directly into gel sample buffer and samples were separated by 12% SDS-PAGE, transferred to nitrocellulose, and reacted with anti-UL3-peptide polyclonal antibody. Bound antibody was detected with [125I]protein A followed by autoradiography. Lanes: M, molecular weight markers; UL3(-Tun), baculovirus-UL3 infected Sf9 cell without tunicamycin treatment; UL3( + Tun), baculovirus-UL3 infected Sf9 cells with tunicamycin treatment; St9, Uninfected cells; Wt., wild-type baculovirus infected Sf9 cells. (b) Localization of baculovirus expressed UL3 in Sf9 cells. Sf9 cells were infected with baculovirus-UL3 as above. The infected cells were harvested, washed with PBS, and suspended in PBS containing 2.5% butanol. The mixture was incubated at room temperature for 5 min and centrifuged at 1000 x g for 10 min. This pellet represents the nuclear/cytoplasmic fraction. The membrane fraction was then pelleted from the original supernatant at 105 000 x g for 1 h at 4°C as described previously [16]. The membranes and cell pellet of baculovirus-UL3 infected Sf9 cells were isolated and analyzed by SDS-PAGE as above. Lanes: M, molecular weight markers; soluble fraction, isolated cell membranes from baculovirus-UL3 infected cells; pellet, nuclear and cytoplasmic fraction from baculovirus-UL3 infected cells. polyclonal antibody. Tunicamycin treatment did n o t significantly alter a n y o f the U L 3 r e l a t e d bands (compare -Tun a n d + T u n lanes, Fig. I(A)), suggesting t h a t the b a c u l o v i r u s - e x p r e s s e d U L 3 d i d n o t c o n t a i n N - l i n k e d oligosaccharides. T h e b a c u l o v i r u s expressed U L 3 p r o t e i n was n o t l a b e l e d b y [3H]mannose i n d i c a t i n g t h a t it d i d n o t c o n t a i n N - l i n k e d o r O - l i n k e d sugars ( d a t a n o t shown). I n a d d i t i o n , b a c u l o v i r u s expressed U L 3 was n o t p r e s e n t on the cell surface (Fig. I(B), soluble fraction) as j u d g e d b y W e s t e r n b l o t a n a l y -

sis following cell f r a c t i o n a t i o n . Thus, U L 3 d i d n o t a p p e a r to be a g l y c o p r o t e i n . T h e b a c u l o v i r u s expressed U L 3 p r o t e i n also d i d n o t a p p e a r to be m y r i s t i l l a t e d , since [14C]myristic acid labeling followed by immunoprecipitation and SDS-PAGE d i d n o t reveal a n y U L 3 r e l a t e d b a n d s ( n o t shown). A n a l y s i s o f the p r e d i c t e d U L 3 a m i n o acid sequence revealed five p o s s i b l e p h o s p h o r y l a t i o n sites. T h r e e linked to serine ( a a 44, 77, 79) a n d two l i n k e d to t h r e o n i n e (aa 36 a n d 48). T o deter-

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Fig. 2. 32pi labeling of baculovirus expressed UL3. Sf9 cells or High Five (HF) cells (BT1-TN-5BI-4, derived from Trichoplusia ni egg cell homogenates) were grown in TNM-FH or Ex-Cell 400 media, respectively(J.R.H. Biosciences, Lenexa, KS). Sf9 cells or HF cells infected with baculovirus-UL3 for 48 h were incubated for 2 h in Hepes buffer pH 7, followed by incubation with 1 mCi/ml of [32p]inorganic orthophosphate or 25 uCi/ml of ~4C-myristicacid (DuPont NEN, Boston, MA) for 5 hr [23]. Cells were harvested, lysed in RIPA buffer, immunoprecipitated using anti-UL3 polyclonal antibody, run on 12% SDS PAGE, treated with Amplify (Americium Life Sciences), dried and exposed for autoradiography. Lanes: M, molecular weight markers; UL3, baculovirus-UL3 infected Sf9 cells; Wt, wild type baculovirus infected Sf9 cells; UL3, baculovirus-UL3 infected HF cells; Wt, wild type baculovirus infected HF cells. mine if the UL3 protein was phosphorylated, baculovirus-UL3 infected Sf9 or H F cells were labeled with 32P i for 5 h at 48 h post infection [23]. The labeled proteins were immunoprecipitated with anti-UL3 antibody and analyzed by SDSP A G E and autoradiography as described above. Baculovirus-UL3 infected cells produced a 32P-labeled band of 33 k D a (Fig. 2, UL3 lanes, arrow). This phosphorylated band was absent in wild-type baculovirus infected cells (Fig. 2, Wt lanes). Thus, the UL3 gene product appeared to be phosphorylated. Mice were immunized three times with baculovirus-UL3 as we previously described for other baculovirus expressed HSV-1 glycoproteins [6] and sera were collected 3 weeks after the final vaccination. Antibodies raised in mice against baculovirus-UL3 did not produce any HSV-1 neutralizing activity (not shown), however, they did recognize the native HSV-1 UL3 protein as judged by Western blots of extracts from HSV-1 infected RS cells. UL3 antibody reacted with

bands of approximately 27 k D a and 33 k D a from extracts of HSV-1 infected RS cells (Fig. 3, lanes - or + tunicamycin; arrows). These bands were not detected in uninfected RS cells (Fig. 3, lane RS cell). Tunicamycin treatment of total cell extracts revealed no changes in mobility of either UL3 related band (Fig. 3, lane + tunicamycin; arrows), indicating that like the baculovirus expressed UL3 protein, the native UL3 protein is not glycosylated. The 27 k D a molecular weight of the smaller band was consistent with size estimates based on the nucleotide sequence of UL3 [20] (lower arrow). The 33 k D a band was similar in size to the phosphorylated band from baculovirus-UL3 infected cells (Fig. 2, upper arrow), suggesting that the native HSV-1 UL3 protein is a phosphoprotein. In contrast to predictions based on sequence analysis, neither the baculovirus expressed UL3 protein or the native HSV-1 UL3 protein appeared to be glycosylated. The UL3 protein was unaffected by tunicamycin treatment and was not

H. Ghiasi et al. / Virus Research 44 (1996) 137-142

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Acknowledgements

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This work was supported by Public Health Service grant EY09224 from the National Eye Institute and The Skirball Program in Molecular Biology.

References 3 0 -~

14.3 ÷ Fig. 3. Antibody raised against baculovirus expressed UL3 reacts with native UL3 from HSV-1 infected RS cells. RS cells were infected with HSV-1 strain KOS at a multiplicity of infection of 10 pfu/cetl for 12 h. Some infected cells were treated with tunicamycin as above. The infected cells were lysed directly into gel sample buffer and samples were separated by 12% SDS-PAGE, transferred to nitrocellulose, and reacted with anti-baculovirus-UL3 polyclonal antibody. Bound antibody was detected with [~25I]protein A followed by autoradiography. Lanes: M, molecular weight markers; Tunicamycin ( - ) , KOS infected RS cells without tunicamycin treatment; Tunicamycin ( + ) , KOS infected RS cells with tunicamycin treatment; RS cells, uninfected rabbit skin cells.

labeled by 3[H]mannose. In contrast, a 33 kDa UL3 related band was labeled by 32Pi, indicating that UL3 was a phosphoprotein. This is consistent with a recent report indicating that the HSV2 UL3 protein is phosphorylated [30]. At least 13 of more than 75 known HSV-1 proteins are phosphorylated [17,20,21]. These include ICP0, ICP4, ICP22, ICP27, gB, gE, and the products of US3, US9, UL13, UL34, UL37, UL41, and UL42 [1,2,5,17,21,22,24,26]. Some of these proteins, such as UL34, require the HSV-1 protein kinase protein (US3) for phosphorylation [26], while others (i.e. UL13) can be phosphorylated without the HSV-I protein kinase [24]. Since the baculovirusUL3 protein was phosphorylated, the HSV-1 UL3 protein did not appear to require other HSV-1 genes in order to become phosphorylated.

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