Herpes Simplex Virus Type 1 Capsid Protein VP26 Interacts with Dynein Light Chains RP3 and Tctex1 and Plays a Role in Retrograde Cellular Transport

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. 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PubMed Scopus Google Scholar). for dynein light chains Tctex1, RP3, and LC8 were from human library HSV-1 genes were from DNA C. A.J. Virology. PubMed Scopus (176) Google Scholar), for which was from by of M. Roizman B. J. Virol. 1998; 72: PubMed Google Scholar). was from P. C. J. Preston V.G. Rixon F.J. J. Gen. Virol. 1994; 75: PubMed Scopus Google Scholar) with and into a were into using a were into for glutathione protein or for protein the yeast two-hybrid genes were into were to and of of heavy chain R.J. J.P. 1998; PubMed Scopus (104) Google Scholar) and HSV-1 R.J. M. E. J. Virol. 2002; 76: PubMed Scopus Google Scholar) have been interactions between dynein subunits and HSV-1 proteins were tested in a yeast two-hybrid R.J. E. M.W. 2002; PubMed Scopus Google Scholar). and of were expressed in with 1 for h at 37 °C. were and R.J. E. M.W. 2002; PubMed Scopus Google Scholar). was expressed a protein at whereas was was R.J. E. M.W. 2002; PubMed Scopus Google Scholar). To were in containing at 4 °C. After 1 h the was at for then the for a was the were with containing and then was into containing with the protein were and at 4 °C. To of was containing and to a of 1 In were expressed in E. with for h at 37 then and R.J. E. M.W. 2002; PubMed Scopus Google Scholar). containing proteins were incubated with of for h with at 4 °C. The were with addition of proteins or HSV-1 were incubated at 4 with were into with at 4 for 2 pull-down assays of dynein was bound to and incubated with cell then bound were R.J. J.P. 1998; PubMed Scopus (104) Google Scholar). Cell were from cells to were with with and then in containing and a were to in a for and at for were by and by R.J. J.P. 1998; PubMed Scopus (104) Google Scholar). used and was also of Recombinant HSV-1 HSV-1 capsids were D.R. Roof L.L. Homa F.L. J. 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PubMed Google of the nucleus was observed but did not involve the nuclear membrane or was used to microtubules. was M. P. J. Virol. 2000; 74: PubMed Scopus Google Scholar), were in with and were and microinjected cells were incubated for 1 h at 37 and M. P. J. Virol. 2000; 74: PubMed Scopus Google Scholar). light chains were detected with of of RP3 S.M. E. S.E. K.T. 1998; PubMed Scopus Google Scholar) or Tctex1 S.M. S.E. R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was used by were were detected using either or J.D. D. D. J. S.M. Rixon F.J. J. Virol. 2002; 76: PubMed Scopus Google Scholar). were using a to a were using the and was using the a where a and a is where not a is of of the of microinjected capsids, the of from the cell nucleus and the of the cell membrane were with the of of A was for that the cell had to whereas the nucleus had to 1 D. M. J. 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PubMed Scopus Google Scholar) was confirmed in our system. dynein subunits in RP3 and Tctex1 not be tested interactions were detected between or LC8 in and of the HSV-1 proteins tested in not The reported interaction between and UL34 not be confirmed UL34 was expressed when in to and in of HSV-1 proteins VP26 and were expressed in E. formed which were by with and then by to 1 VP26 expressed in and then has been shown to to HSV-1 capsids, in either or 1 S. J. of in the of Herpes 1 (HSV-1) of Scholar, D.R. Homa F.L. J. Virol. 1997; PubMed Google Scholar). was expressed at In vitro pull-down assays were by HSV-1 proteins with of dynein subunits DIC, RP3, and Tctex1, bound to and from were confirmed for with at the bound to and but not to or and was not mediated the a heavy chain did not to RP3 or Tctex1 There was evidence of dynein binding to but this not to in that VP26 binds to dynein light chains in a dynein complex, well in an in vitro pull-down of cytoplasmic dynein was of HSV-1 proteins VP26 and were bound to and incubated with from and the were by and which were detected in cell bound to but not to The of in the bound complex was used a for intact dynein we have evidence of a interaction between VP26 and in of and in was confirmed by Recombinant HSV-1 VP26 to investigate the of capsid proteins in and recombinant HSV-1 capsids were constructed with or without VP26 or capsids were by and shown to be in not were used in pull-down assays with dynein subunits bound to The or of VP26 was confirmed by and A and VP26+ capsids bound to but not to dynein in VP26-capsids did not in either The of capsids bound to of the dynein chains was detected using the major capsid protein but not VP26-capsids in Recombinant HSV-1 capsids were incubated with dynein bound to in a pull-down bound to proteins were detected by using in and VP26+ capsids bound to but not to dynein of were also VP26-capsids did not Recombinant HSV-1 with RP3, Tctex1, and in an in interaction between HSV-1 capsids and cytoplasmic dynein, cells were on and microinjected with a of recombinant HSV-1 capsids were after 1 h at 37 and for capsids well for either dynein light chains or or microtubules the of capsids cell and of capsids with dynein light chains RP3 and Tctex1 well with microtubules using the confirmed with dynein and microtubules for the of HSV-1 capsids and RP3 and Tctex1 were also in the of cells not Recombinant HSV-1 VP26 the in a for the interaction between VP26 and dynein, cells were on and microinjected with a of recombinant HSV-1 capsids or were then either or after 2 or 4 h with and the of capsids cell by and VP26+ capsids were the A and After 2 h at 37 VP26-capsids remained in a whereas VP26+ capsids had the nuclear in cells were observed after 4 h not To the of capsids at a was to that the membrane to whereas the cell nucleus to 1 a were in observed the nucleus of and cells were of and were not There was of the nuclear membrane, and to the nuclear membrane be were used to investigate the from VP26+ capsids, the on by 2 or 4 the nucleus In VP26-capsids the on by results that the binding of HSV-1 outer capsid protein VP26 to dynein light chains RP3, and Tctex1, is in the retrograde transport of viral capsids the cell nucleus during infection. Cytoplasmic dynein is the major molecular and dynein complex of the 14-kDa light either RP3 or Tctex1. The homologous at the S.M. E. S.E. K.T. 1998; PubMed Scopus Google Scholar), are in complex, for binding on DIC, and have binding J. Cell Biol. 2001; PubMed Scopus (104) Google Scholar). We binding of VP26 to dynein light chains RP3 and Tctex1 in a yeast two-hybrid and we confirmed strong of the by work confirmed binding of VP26 to RP3 and Tctex1 in using and pull-down We also confirmed that is to to intact cytoplasmic dynein in cell we have confirmed binding of dynein to VP26 in its of a recombinant viral capsid bound to we have confirmed binding of VP26 to dynein light chain RP3 in of a viral that the binding of VP26 to RP3 VP26 from involved in binding to in capsids P. S. J. Virol. 2003; PubMed Scopus Google Scholar) and is with the interaction a role in Furthermore, in microinjected cells we have shown of HSV-1 capsids containing VP26 with RP3, Tctex1, and a role for Despite strong interaction between VP26 and Tctex1 in the yeast two-hybrid and in vitro pull-down assays and of HSV-1 capsids with Tctex1, we were to binding of VP26+ capsids to Tctex1 in We that this most likely a for this in light of the in vitro binding The to either RP3 or Tctex1 be to HSV-1 an by retrograde transport of HSV-1 capsids on cytoplasmic dynein complex in all cell We that recombinant Tctex1 to VP26 in vitro and not to VP26 into recombinant HSV-1 the of VP26+ capsids with Tctex1 in live cells that VP26 interacts with Tctex1, well RP3, in this model. HSV-1 infects mucous in adults and being transported in a retrograde direction along neuronal axons to latent infection. RP3 in is an to mediate this retrograde transport, it is expressed at in cells by HSV-1, and whereas Tctex1 is expressed in and S.M. E. S.E. K.T. 1998; PubMed Scopus Google Scholar). Furthermore, it has been that dynein the role during retrograde transport from nerve J. 2001; PubMed Google Scholar), the site of HSV-1 at the is to interact with molecular D.R. Homa F.L. J. Virol. 1997; PubMed Google Scholar), most of the tegument is after cell (6Ojala P.M. Sodeik B. Ebersold M.W. Kutay U. Helenius A. Mol. Cell. Biol. 2000; 20: 4922-4931Crossref PubMed Scopus (203) Google Scholar). proteins and are to dissociate from the virus at of infection, whereas the major tegument protein and the capsid protein to incoming capsids (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, 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, M. J. Virol. 2002; 76: PubMed Scopus Google Scholar, P. J. Virol. PubMed Google Scholar). to to capsids at the J. Rixon F.J. J. Virol. PubMed Google Scholar), whereas VP26 is on Homa F.L. D.R. N. J. Virol. PubMed Google Scholar, D.R. Homa F.L. J. Virol. 1997; PubMed Google Scholar, J. J. J.D. Rixon F.J. Biol. 2: PubMed Scopus Google Scholar) and does not with interaction J. D. J. Zhou M. Rixon F.J. J. Virol. 2001; 75: PubMed Scopus Google Scholar). binding is to of the of VP5, the of which has recently been Rixon F.J. J. 2003; PubMed Scopus Google Scholar). of the tegument protein to RP3 and Tctex1 was detected in the yeast two-hybrid but not be confirmed with pull-down assays to of proteins in E. The of results is but role is likely to be in the viral during retrograde that is involved in retrograde transport of the complex, it from incoming capsids during cell infection M. J. Virol. 2002; 76: PubMed Scopus Google Scholar), of during retrograde transport be to interaction of with dynein a role during tegument newly has been observed in M. J. Virol. 2002; 76: PubMed Scopus Google Scholar). To that the interaction between VP26 and dynein is for cellular transport, recombinant viral capsids were microinjected into living was of VP26+ capsids the cell nucleus after 2 or 4 which was absent for VP26-capsids in the cell but this to a distribution. results an role for VP26 during retrograde transport, through interactions with cytoplasmic The of tegument or capsid proteins to HSV-1 transport is and We that or of the capsid proteins also contribute to dynein binding in by 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, M.J. Smith G.A. Enquist L.W. Traffic. 2001; 2: 429-436Crossref PubMed Scopus (104) Google Scholar). binding of or HSV-1 proteins to dynein in viral proteins for retrograde transport, and VP26 is not an protein for in vitro P. S. Virology. 1998; PubMed Scopus Google Scholar). for an be for the virus, and are for in HSV-1 proteins A. J. Virol. 2003; PubMed Scopus Google Scholar). In our yeast two-hybrid was evidence of interaction between dynein and a large of but it was not to the of the was it to in our for interaction with RP3 or Tctex1, to The role of VP26 during viral transport in to be There has been in a the role of VP26 in retrograde transport of HSV-1 and P. S. Virology. 1998; PubMed Scopus Google Scholar). of VP26 the of virus in by whereas in cell retrograde transport is less were deletion of the of VP26 deletion at h after infection, the virus be transported in the of VP26. be to a in transport or virus can h Knipe D.M. D.M. J. Virol. 1998; 72: PubMed Google Scholar). In our we to the viral and cellular proteins that mediate retrograde axonal transport of HSV-1. of interaction assays and recombinant capsids be a to interactions on which can interactions where or proteins (10Tomishima M.J. Smith G.A. Enquist L.W. Traffic. 2001; 2: 429-436Crossref PubMed Scopus (104) Google Scholar). We have by assays that VP26 binds to dynein light chains RP3 and Tctex1 in and we confirmed binding of VP26 to intact dynein complexes. We have in cells of HSV-1 capsids with RP3, Tctex1, and microtubules. We have shown that VP26 in its is to mediate binding of recombinant HSV-1 capsids to RP3 in in the of capsid or tegument proteins. we have shown that into recombinant capsids, is and sufficient to mediate retrograde intracellular transport of We propose that not for viral in is likely to be of or proteins retrograde axonal transport in of for viral We for with the yeast two-hybrid system. We also and for with the capsid