Title: Herpes Simplex Virus Type 1 Capsid Protein VP26 Interacts with Dynein Light Chains RP3 and Tctex1 and Plays a Role in Retrograde Cellular Transport
Abstract: Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 °C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 °C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains. Herpes simplex virus type 1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus type 1; DIC, dynein intermediate chain; PRV, pseudorabies virus; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 1The abbreviations used are: HSV-1, herpes simplex virus type 1; DIC, dynein intermediate chain; PRV, pseudorabies virus; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. infects 40–80% of people worldwide and can cause potentially fatal meningoencephalitis in adults or disseminated infection in neonates, in addition to common mucocutaneous disease. After inoculation of the skin or mucous membrane, HSV-1 is transported along sensory axons in a retrograde direction to the neuronal cell body, where it establishes life-long latent infection. Periodic reactivation results in HSV-1 being transported in an anterograde direction to nerve terminals, where it causes recurrent clinical disease or asymptomatic viral shedding (1Roizman B. Sears A.E. Fields B.N. Knipe D.M. Howley P.M. Chanock R.M. Melnick J.L. Monath T.P. Roizman B. Straus S.E. Fields Virology. 3rd Ed. Lippincott-Raven, Philadelphia, PA1996: 2231-2294Google Scholar). The double-stranded DNA virus HSV-1 has a 1250-Å icosahedral protein capsid, surrounded by a less structured protein tegument layer, in turn surrounded by a lipid envelope containing several glycoproteins. The major capsid proteins VP5, VP19C, VP23, and VP26 are self-assembling when expressed in vitro using recombinant baculoviruses (2Tatman J.D. Preston V.G. Nicholson P. Elliott R.M. Rixon F.J. J. Gen. Virol. 1994; 75: 1101-1113Crossref PubMed Scopus (155) Google Scholar, 3Thomsen D.R. Roof L.L. Homa F.L. J. Virol. 1994; 68: 2442-2457Crossref PubMed Google Scholar). During infection, HSV-1 binds to cell surface receptors (via glycoproteins), enters the cell by membrane fusion, then most but not all tegument proteins dissociate from the nucleocapsid after phosphorylation (4Morrison E.E. Stevenson A.J. Wang Y.F. Meredith D.M. J. Gen. Virol. 1998; 79: 2517-2528Crossref PubMed Scopus (69) Google Scholar, 5Morrison E.E. Wang Y.F. Meredith D.M. J. Virol. 1998; 72: 7108-7114Crossref PubMed Google Scholar). The nucleocapsid-tegument complex is transported to the outer nuclear membrane where it docks and releases viral DNA into the nucleus, but the capsid itself does not enter the nucleus (6Ojala P.M. Sodeik B. Ebersold M.W. Kutay U. Helenius A. Mol. Cell. Biol. 2000; 20: 4922-4931Crossref PubMed Scopus (203) Google Scholar). There is evidence that the rapid "retrograde" transport of this complex to the cell nucleus involves microtubules and is mediated by the minus-end-directed molecular motor cytoplasmic dynein (7Sodeik B. Ebersold M.W. Helenius A. J. Cell Biol. 1997; 136: 1007-1021Crossref PubMed Scopus (549) Google Scholar, 8Dohner K. Wolfstein A. Prank U. Echeverri C. Dujardin D. Vallee R. Sodeik B. Mol. Biol. Cell. 2002; 13: 2795-2809Crossref PubMed Scopus (257) Google Scholar, 9Lycke E. Kristensson K. Svennerholm B. Vahlne A. Ziegler R. J. Gen. Virol. 1984; 65: 55-64Crossref PubMed Scopus (96) Google Scholar). Retrograde transport of the closely related alphaherpesvirus pseudorabies virus (PRV) has been observed in live cells to be saltatory, with brief, rapid transport events (10Tomishima M.J. Smith G.A. Enquist L.W. Traffic. 2001; 2: 429-436Crossref PubMed Scopus (104) Google Scholar). The herpes viral proteins that mediate retrograde transport are unknown but are likely to involve outer capsid or inner tegument proteins. In support of this, HSV-1 capsids, stripped of envelope and much of the tegument protein by detergent lysis, move in a retrograde direction after injection into giant squid axons (11Bearer E.L. Breakefield X.O. Schuback D. Reese T.S. LaVail J.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8146-8150Crossref PubMed Scopus (116) Google Scholar). The site of attachment for the dynein complex on the capsid has not been confirmed. Cytoplasmic dynein is a large (1.2 MDa) complex, with heavy chains providing motive force, whereas intermediate and light chains contribute to cargo binding (12King S.M. Biochim. Biophys. Acta. 2000; 1496: 60-75Crossref PubMed Scopus (289) Google Scholar). Regulation of dynein function is not well understood but is thought to involve the multisubunit complex dynactin, which is also involved in binding membranous cargo (13Holleran E.A. Karki S. Holzbaur E.L. Int. Rev. Cytol. 1998; 182: 69-109Crossref PubMed Google Scholar). Light chain LC8 has been reported to interact with proteins from rabies virus (14Raux H. Flamand A. Blondel D. J. Virol. 2000; 74: 10212-10216Crossref PubMed Scopus (259) Google Scholar, 15Jacob Y. Badrane H. Ceccaldi P.E. Tordo N. J. Virol. 2000; 74: 10217-10222Crossref PubMed Scopus (198) Google Scholar), African swine fever virus (16Alonso C. Miskin J. Hernaez B. Fernandez-Zapatero P. Soto L. Canto C. Rodriguez-Crespo I. Dixon L. Escribano J.M. J. Virol. 2001; 75: 9819-9827Crossref PubMed Scopus (144) Google Scholar), human adenovirus, vaccinia virus, and human papillomavirus (17Martinez-Moreno M. Navarro-Lerida I. Roncal F. Albar J.P. Alonso C. Gavilanes F. Rodriguez-Crespo I. FEBS Lett. 2003; 544: 262-267Crossref PubMed Scopus (64) Google Scholar), whereas Tctex1 interacts with the poliovirus receptor CD155 (18Mueller S. Cao X. Welker R. Wimmer E. J. Biol. Chem. 2002; 277: 7897-7904Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Despite a recently reported interaction between dynein intermediate chain (DIC) and HSV-1 protein UL34 (19Ye G.J. Vaughan K.T. Vallee R.B. Roizman B. J. Virol. 2000; 74: 1355-1363Crossref PubMed Scopus (143) Google Scholar), its role in retrograde transport has yet to be confirmed. The protein product of UL34 is absent from mature virions in HSV-1 (20Reynolds A.E. Wills E.G. Roller R.J. Ryckman B.J. Baines J.D. J. Virol. 2002; 76: 8939-8952Crossref PubMed Scopus (277) Google Scholar) and PRV (21Klupp B.G. Granzow H. Mettenleiter T.C. J. Virol. 2000; 74: 10063-10073Crossref PubMed Scopus (168) Google Scholar). Furthermore, deletion of UL34 from HSV-1 does not prevent infection of cells (22Roller R.J. Zhou Y. Schnetzer R. Ferguson J. DeSalvo D. J. Virol. 2000; 74: 117-129Crossref PubMed Scopus (176) Google Scholar). Previous work in our laboratory has concentrated on anterograde axonal transport of HSV-1. We have shown that newly formed HSV-1 capsids, having acquired much of their tegument in the neuron cell body (23Miranda-Saksena M. Boadle R.A. Armati P. Cunningham A.L. J. Virol. 2002; 76: 9934-9951Crossref PubMed Scopus (56) Google Scholar), are transported in an anterograde direction along axons, separate from glycoproteins (24Holland D.J. Miranda-Saksena M. Boadle R.A. Armati P. Cunningham A.L. J. Virol. 1999; 73: 8503-8511Crossref PubMed Google Scholar, 25Miranda-Saksena M. Armati P. Boadle R.A. Holland D.J. Cunningham A.L. J. Virol. 2000; 74: 1827-1839Crossref PubMed Scopus (112) Google Scholar). Similar observations have been made for PRV (26Enquist L.W. Tomishima M.J. Gross S. Smith G.A. Vet. Microbiol. 2002; 86: 5-16Crossref PubMed Scopus (73) Google Scholar). Fast anterograde, microtubule-dependent transport is mediated by the kinesin family of molecular motors (27Goldstein L.S. Yang Z. Annu. Rev. Neurosci. 2000; 23: 39-71Crossref PubMed Scopus (448) Google Scholar). We have shown previously (28Diefenbach R.J. Miranda-Saksena M. Diefenbach E. Holland D.J. Boadle R.A. Armati P.J. Cunningham A.L. J. Virol. 2002; 76: 3282-3291Crossref PubMed Scopus (121) Google Scholar) that the HSV-1 tegument protein US11 interacts with the ubiquitous heavy chain of the kinesin motor KIF5B and is likely to play an important role in anterograde axonal transport. By using a similar approach for retrograde transport, we report an interaction between the HSV-1 capsid protein VP26 and 14-kDa dynein light chains RP3 and Tctex1. These light chains are 55% homologous at the amino acid level and are mutually exclusive in cytoplasmic dynein complexes (29Chuang J.Z. Milner T.A. Sung C.H. J. Neurosci. 2001; 21: 5501-5512Crossref PubMed Google Scholar, 30King S.M. Barbarese E. Dillman III, J.F. Benashski S.E. Do K.T. Patel-King R.S. Pfister K.K. Biochemistry. 1998; 37: 15033-15041Crossref PubMed Scopus (97) Google Scholar, 31Tai A.W. Chuang J.Z. Sung C.H. J. Cell Biol. 2001; 153: 1499-1509Crossref PubMed Scopus (104) Google Scholar). We propose that VP26 mediates binding of the HSV-1 nucleocapsid to cytoplasmic dynein, via interactions with RP3 and probably Tctex1, during retrograde axonal transport of virus in neurons as well as during infection of non-neuronal cells. Expression Constructs—Genes were amplified by PCR using Geneamp® XL PCR Kit (Applied Biosystems). The entire open reading frame for each gene was cloned into recombinant expression vectors in all cases except for HSV-1 UL36, where the N-terminal two-thirds of the gene (amino acids 1–1874) were cloned. Oligonucleotide primers, incorporating EcoRI and/or XhoI restriction endonuclease sites for most constructs, were designed using Primer3 software (32Rozen S. Skaletsky H.J. Krawetz S. Misener S. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press Inc., Totowa, NJ2000: 365-386Google Scholar) and BioManager by ANGIS (www.angis.org.au). For UL36 a 3′ BglII site was used for insertion into BamHI sites, and for UL32 NcoI sites were used. Human DIC-1c was amplified from a pBluescript plasmid, kindly provided by Dr. Lap-Chee Tsui, University of Toronto (34Crackower M.A. Sinasac D.S. Xia J. Motoyama J. Prochazka M. Rommens J.M. Scherer S.W. Tsui L.C. Genomics. 1999; 55: 257-267Crossref PubMed Scopus (35) Google Scholar). Genes for dynein light chains Tctex1, RP3, and LC8 were amplified from human brain cDNA library (Display Biosystems Biotech). HSV-1 genes were amplified from overlapping DNA cosmids encoding strain 17 (35Cunningham C. Davison A.J. Virology. 1993; 197: 116-124Crossref PubMed Scopus (176) Google Scholar), except for US11 which was amplified from plasmid pRB4766 (strain 17), kindly provided by Bernard Roizman, University of Chicago (36Cassady K.A. Gross M. Roizman B. J. Virol. 1998; 72: 8620-8626Crossref PubMed Google Scholar). UL19 was excised from plasmid pE19 (33Nicholson P. Addison C. Cross A.M. Kennard J. Preston V.G. Rixon F.J. J. Gen. Virol. 1994; 75: 1091-1099Crossref PubMed Scopus (74) Google Scholar) with BglII and inserted into a BamHI site. Digested PCR products were inserted into expression plasmids using a Clonables™ kit (Novagen). Genes were cloned into pGEX-5X-1 (Amersham Biosciences) for glutathione S-transferase (GST) tag fusion protein expression or pET-28a (Novagen) for hexahistidine (His6) tag fusion protein expression. For the LexA yeast two-hybrid system genes were cloned into displayBait and/or displayTarget vectors (Display Systems Biotech). All constructs were sequenced to confirm gene sequence and correct reading frame. Construction of His6 tag fusions of kinesin heavy chain KIF5B (amino acids 771–963) (37Diefenbach R.J. Mackay J.P. Armati P.J. Cunningham A.L. Biochemistry. 1998; 37: 16663-16670Crossref PubMed Scopus (104) Google Scholar) and HSV-1 US11 (28Diefenbach R.J. Miranda-Saksena M. Diefenbach E. Holland D.J. Boadle R.A. Armati P.J. Cunningham A.L. J. Virol. 2002; 76: 3282-3291Crossref PubMed Scopus (121) Google Scholar) have been described previously. LexA Yeast Two-hybrid Assay—Potential interactions between dynein subunits and HSV-1 proteins were tested in a Clontech Matchmaker LexA yeast two-hybrid system as described previously (38Diefenbach R.J. Diefenbach E. Douglas M.W. Cunningham A.L. Biochemistry. 2002; 41: 14906-14915Crossref PubMed Scopus (45) Google Scholar). Expression and Purification of His6-tagged Proteins—His6 tag constructs were expressed in Escherichia coli BL21 (DE3), induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C. Proteins were harvested and lysed as described previously (38Diefenbach R.J. Diefenbach E. Douglas M.W. Cunningham A.L. Biochemistry. 2002; 41: 14906-14915Crossref PubMed Scopus (45) Google Scholar). His-VP11/12 was expressed as a soluble protein at low levels, whereas His6-VP26 was insoluble (i.e. present only as inclusion bodies). His6-KIF5B was purified as described previously (38Diefenbach R.J. Diefenbach E. Douglas M.W. Cunningham A.L. Biochemistry. 2002; 41: 14906-14915Crossref PubMed Scopus (45) Google Scholar). To solubilize His6-VP26, inclusion bodies were resuspended in "denaturing buffer" (8 m urea, 100 mm NaH2PO4, 10 mm Tris-HCl, pH 8.0) containing 0.5 mm imidazole at 4 °C. After 1 h the solution was centrifuged at 16,000 × g for 30 min, then the supernatant for a further 10 min. Supernatant was passed over nickel beads (His.Bind® kit, Novagen); the beads were washed with denaturing buffer containing 20 mm imidazole, and then His6-VP26 was eluted into denaturing buffer containing 200 mm imidazole. Fractions with the highest protein concentration (∼5 mg/ml) were pooled and stored at 4 °C. To refold His6-VP26, 200 μl of stock His6-VP26 solution (in 8 m urea) was slowly diluted 1:8 (in buffer containing 100 mm NaH2PO4 and 10 mm Tris-HCl, pH 8.0) to a final urea concentration of 1 m. In Vitro Pull-down Assays—GST fusion tag constructs were expressed in E. coli BL21, induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C, then harvested, and lysed as described previously (38Diefenbach R.J. Diefenbach E. Douglas M.W. Cunningham A.L. Biochemistry. 2002; 41: 14906-14915Crossref PubMed Scopus (45) Google Scholar). Soluble bacterial lysates (1 ml) containing GST fusion proteins were incubated with 50 μl of glutathione-Sepharose beads (Amersham Biosciences) for 3 h with rocking at 4 °C. The beads were washed three times with lysis buffer (minus protease inhibitors) before addition of purified His6 tag fusion proteins or HSV-1 capsids. Beads were incubated overnight at 4 °C with rocking before washing five times as above. Protein complexes were eluted into buffer (50 mm Tris-HCl, pH 8.0, 10 mm reduced glutathione) with rocking at 4 °C for 2 h. For pull-down assays of dynein complexes, refolded His6-VP26 (prepared as above) was bound to nickel beads (His.Bind® kit, Novagen) and incubated with cell lysates; then bound complexes were eluted as described previously (37Diefenbach R.J. Mackay J.P. Armati P.J. Cunningham A.L. Biochemistry. 1998; 37: 16663-16670Crossref PubMed Scopus (104) Google Scholar). Cell lysates were prepared from Hep2 cells grown to 80% confluence. Cells were detached with trypsin, washed with phosphate-buffered saline, and then resuspended in lysis buffer containing phosphate-buffered saline, 0.1% Triton X-100, and a protease inhibitor mixture (Sigma). Cells were subjected to sonication in a cup-horn water bath (three times for 20 s, 100% duty cycle) and centrifuged at 12,000 × g for 30 min. Western Blots—Proteins were separated by SDS-PAGE and identified by immunoblotting as described previously (37Diefenbach R.J. Mackay J.P. Armati P.J. Cunningham A.L. Biochemistry. 1998; 37: 16663-16670Crossref PubMed Scopus (104) Google Scholar). Antibodies used included mouse monoclonal antibodies (Santa Cruz Biotechnology) against fusion tags hemagglutinin, LexA, and His6. Monoclonal antibody against DIC (Chemicon) was also used. Preparation of Recombinant HSV-1 Capsids—Recombinant HSV-1 capsids were prepared as described previously (3Thomsen D.R. Roof L.L. Homa F.L. J. Virol. 1994; 68: 2442-2457Crossref PubMed Google Scholar). Briefly, recombinant baculoviruses were used to express either five or six HSV-1 capsid genes (UL18, UL19, UL26, UL26.5, and UL38, with or without UL35) in insect Sf9 cells. The resulting capsids, purified by sucrose gradient centrifugation, have the same morphology as HSV B capsids (3Thomsen D.R. Roof L.L. Homa F.L. J. Virol. 1994; 68: 2442-2457Crossref PubMed Google Scholar, 39Trus B.L. Homa F.L. Booy F.P. Newcomb W.W. Thomsen D.R. Cheng N. Brown J.C. Steven A.C. J. Virol. 1995; 69: 7362-7366Crossref PubMed Google Scholar). All recombinant capsids contain major capsid proteins VP5, VP19C, and VP23 and scaffolding proteins VP21, VP22a, and VP24. Capsids formed in the presence of UL35 also contain capsid protein VP26. For microinjection, capsids were diluted 1:10 in 20 mm Tris-HCl buffer, pH 7.5, containing 0.5 m NaCl and 1 mm EDTA, subjected to sonication in a cup-horn water bath (three times for 40 s, 50% duty cycle), and centrifuged at 12,000 × g for 30 s. The approximate final protein concentration, measured by Bio-Rad Protein Assay, was 0.1 mg/ml. Microinjection of Recombinant HSV-1 Capsids—Hep2 cells were grown at 37 °C (5% CO2) on CELLocate glass coverslips (Eppendorf) in Dulbecco's modified Eagle's medium (Invitrogen) with 9% fetal calf serum (JRH Bioscience). Microinjection was performed at room temperature, using Dulbecco's modified Eagle's medium with 2% fetal calf serum, buffered with 25 mm HEPES-NaOH, pH 7.4. For co-localization experiments PTK2 cells were grown in minimum Eagle's medium (Invitrogen) supplemented with nonessential amino acids (0.1 mm), sodium pyruvate (1 mm), and 10% fetal calf serum (JRH Biosciences). An Eppendorf FemtoJet® microinjector and InjectMan® NI2 micromanipulator, attached to a Nikon DM-IRB inverted microscope, were used to inject recombinant HSV-1 capsids through a Femtotip® glass micropipette into Hep2 or PTK2 cells. The injection parameters used are as follows: compensation pressure, 50 hPa; injection pressure, 280–300 hPa (range 200–400 hPa); injection time, 0.2–0.4 s. Between 200 and 400 cells were injected for each experimental group or time point. Immunofluorescence and Confocal Microscopy—Microinjected Hep2 cells were fixed and permeabilized as described previously (25Miranda-Saksena M. Armati P. Boadle R.A. Holland D.J. Cunningham A.L. J. Virol. 2000; 74: 1827-1839Crossref PubMed Scopus (112) Google Scholar), either immediately or after 2–4 h of incubation at 37 °C. Sensitive and specific labeling of intra-cytoplasmic capsids was obtained with rabbit polyclonal anti-VP5 antibody (NC1, kindly provided by Dr. G. Cohen and Dr. R. Eisenberg, University of Pennsylvania, Philadelphia (40Cohen G.H. Ponce D.L. Diggelmann H. Lawrence W.C. Vernon S.K. Eisenberg R.J. J. Virol. 1980; 34: 521-531Crossref PubMed Google Scholar)). Some nonspecific labeling of antigens within the nucleus was observed but did not involve the nuclear membrane or cytoplasm. Mouse anti-bovine α-tubulin antibody (monoclonal antibody 236-10501, Molecular Probes) was used to label microtubules. Immunolabeling was as described previously (25Miranda-Saksena M. Armati P. Boadle R.A. Holland D.J. Cunningham A.L. J. Virol. 2000; 74: 1827-1839Crossref PubMed Scopus (112) Google Scholar), except antibodies were diluted in Tris-buffered saline with goat serum (1.5% v/v), bovine serum albumin (0.1% w/v), Tween 20 (0.05% v/v), and NaNH3 (0.02 m). Secondary antibodies were Cy™ 3-conjugated goat anti-mouse IgG (Amersham Biosciences) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Molecular Probes). For co-localization experiments, microinjected PTK2 cells were incubated for 1 h at 37 °C, fixed, and permeabilized (25Miranda-Saksena M. Armati P. Boadle R.A. Holland D.J. Cunningham A.L. J. Virol. 2000; 74: 1827-1839Crossref PubMed Scopus (112) Google Scholar). Dynein light chains were detected with rabbit polyclonal antibodies (a kind gift of Dr. Stephen King, University of Connecticut Health Centre, Farmington, CT). Primary antibody R5270 against RP3 (30King S.M. Barbarese E. Dillman III, J.F. Benashski S.E. Do K.T. Patel-King R.S. Pfister K.K. Biochemistry. 1998; 37: 15033-15041Crossref PubMed Scopus (97) Google Scholar) or R5205 against Tctex1 (41King S.M. Dillman III, J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem. 1996; 271: 32281-32287Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) was used as above, followed by Cy™ 3-conjugated goat anti-rabbit IgG (Amersham Biosciences). Microtubules were labeled as above. Capsids were detected using anti-VP5 antibodies, either rabbit polyclonal (NC1, as above) or mouse monoclonal antibody (DM165, raised against purified VP5) (42McClelland D.A. Aitken J.D. Bhella D. McNab D. Mitchell J. Kelly S.M. Price N.C. Rixon F.J. J. Virol. 2002; 76: 7407-7417Crossref PubMed Scopus (31) Google Scholar). Confocal images were acquired using a Leica TCS SP2 Confocal System (software version 2.00 Build 0871), attached to a Leica DMRE microscope. Dual-channel images were overlaid using the Leica software, and co-localization was determined using the Boolean "AND" operator. For a red/green overlay, where a red and green pixel colocalize, a bright pixel is displayed; where pixels do not co-localize, a black pixel is displayed. Measurement of Intracellular Distribution of Capsids—To assess quantitatively the distribution of microinjected capsids, the distances of each fluorescent particle from the cell nucleus a, and the nearest part of the cell membrane b, were measured with the aid of image analysis software (ImageJ version 1.29, National Institutes of Health). A "Nuclear Migration Index" b/(a + b) was calculated for each particle, such that particles near the cell periphery had values close to 0, whereas particles near the nucleus had values close to 1 (43McDonald D. Vodicka M.A. Lucero G. Svitkina T.M. Borisy G.G. Emerman M. Hope T.J. J. Cell Biol. 2002; 159: 441-452Crossref PubMed Scopus (634) Google Scholar). Image analysis was performed "blinded" to remove potential observer bias. Fluorescent particles were confirmed to be within the cytoplasm, rather than sitting on the cell membrane, using serial z sections. In areas where several fluorescent particles congregated, individual particles were visually separated by image analysis using threshold functions or z sections. Statistical Analysis—The statistical software package SPSS for Windows (version 11.0) was used to analyze the data. For comparison of quantitative β-galactosidase activity in the yeast two-hybrid system, analysis of variance was performed. Tukey's correction for multiple comparisons was then used to investigate pairwise differences between Target (dynein) inserts for each Bait (HSV) insert. To investigate formally intracellular movement of capsids in microinjection experiments, general linear models were used, with experiment as a random effect and time and VP26 status as fixed effects. VP26 and VP11/12 Bind to RP3 and Tctex1 in the Yeast Two-hybrid System—A yeast two-hybrid matrix approach was used to screen for interactions between HSV-1 capsid or tegument proteins and cytoplasmic dynein. Genes for dynein subunits DIC, LC8, RP3, and Tctex1, as well as for HSV-1 capsid and tegument proteins, were cloned into both Bait vector (fusion construct with LexA DNA binding domain), and Target vector (fusion construct with B42 DNA activation domain). The proteins were tested pairwise for interactions in all available combinations. A strong interaction was indicated by both growth on media lacking leucine and a blue color change of yeast colonies within 48 h. Interactions were further confirmed with a quantitative β-galactosidase assay. Known interactions between DIC and dynein light chains (31Tai A.W. Chuang J.Z. Sung C.H. J. Cell Biol. 2001; 153: 1499-1509Crossref PubMed Scopus (104) Google Scholar, 44Mok Y.K. Lo K.W. Zhang M. J. Biol. Chem. 2001; 276: 14067-14074Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 45Makokha M. Hare M. Li M. Hays T. Barbar E. Biochemistry. 2002; 41: 4302-4311Crossref PubMed Scopus (86) Google Scholar), as well as LC8 dimerization, were confirmed in our system (Table I).Table IYeast two-hybrid screen HSV-1 proteins, inserted in Bait vector, were tested for interaction with dynein subunits and inserted in Target vector in a LexA yeast two-hybrid system. "Positive interactions," defined as growth on leucine-deficient media and blue color change of yeast colonies within 48 h, were confirmed using a quantitative β-galactosidase assay (shown in arbitrary units). The symbols used are as follows: +, 10–50; ++, 20–300; +++, 300–1000; ++++, >1000; -, no color change at 48 h; (-), no color change but protein was poorly expressed; AA, strong auto-activation at 48 h; ND, not determined due to autoactivation; (-)ve, display Target with no protein insert.BaitTarget(–)veDICLC8RP3Tctex1UL6UL38DIC––+++++++++++––LC8–++++++––––US11–––––UL6–––––UL13(–)(–)(–)(–)(–)UL17AANDNDNDNDNDNDUL18 (VP23)––––––+++UL19 (VP5)(–)(–)(–)(–)(–)(–)(–)UL25AANDNDNDNDNDNDUL32AANDNDNDNDNDNDUL35 (VP26)–+++++++++++–UL36aUL36 amino acids 1–1874 (VP1/2)–––––UL37–––––UL38 (VP19C)AANDNDNDNDNDNDUL41 (VHS)–––––UL46 (VP11/12)–++++++++++++–UL47 (VP13/14)–––––UL48 (VP16)AANDNDNDNDNDNDa UL36 amino acids 1–1874 Open table in a new tab With HSV-1 proteins in Bait vector a strong interaction was detected between VP26 (UL35) and VP11/12 (UL46) with homologous 14-kDa dynein light chains RP3 and Tctex1 (Table I). β-Galactosidase activity for each protein interaction was measured using a quantitative assay and statistically examined using analysis of variance (Fig. 1). β-Galactosidase activity was significantly greater for interactions with RP3 or Tctex1 than for DIC (p < 0.05), LC8 (p < 0.004), or the negative control (p < 0.003). The apparent small increase for interactions with DIC and LC8 was not statistically significant. The known interaction between HSV-1 capsid proteins VP23 and VP19C (46Desai P. Person S. Virology. 1996; 220: 516-521Crossref PubMed Scopus (54) Google Scholar) was confirmed in our system. With dynein subunits in Bait vector, RP3 and Tctex1 strongly auto-activated, so they could not be tested further. No interactions were detected between DIC or LC8 in Bait vector and any of the HSV-1 proteins tested in Target vector (data not shown). The previously reported interaction between DIC and UL34 could not be confirmed because UL34 was poorly expressed when inserted in Target vector. His6-VP26 Binds to GST-RP3 and GST-Tctex1 in Vitro—His6 fusion constructs of HSV-1 proteins VP26 and VP11/12 were expressed in E. coli. His6-VP26 formed insoluble inclusion bodies, which were solubilized by denaturing with 8 m urea and then slowly refolded by dilution to 1 m urea. VP26 expressed in bacteria, denatured, and then refolded has been shown previously to bind to HSV-1 capsids, in either CHAPS buffer or 1 m urea (47.Macnab-Bain, S. J. (1999) Analysis of Protein-Protein Interactions in the Shell of Herpes Simplex Virus Type 1 (HSV-1) Caspids. Ph.D. thesis,