Identification of a Novel Signal Sequence That Targets Transmembrane Proteins to the Nuclear Envelope Inner Membrane

Abstract

Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble β-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885–890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins. Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble β-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885–890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins. rough endoplasmic reticulum endoplasmic reticulum inner nuclear membrane lamin B receptor lamina-associated polypeptide 1 and 2, respectively glycoprotein B herpes simplex virus type 1 human cytomegalovirus nuclear localization signal polymerase chain reaction monoclonal antibody phosphate-buffered saline polyacrylamide gel electrophoresis amino acid(s) The nuclear envelope consists of three morphologically distinct membrane domains (1.Gerace L. Burke B. Annu. Rev. Cell Biol. 1988; 4: 335-374Crossref PubMed Scopus (519) Google Scholar, 2.Goldberg M.W. Allen T.D. Curr. Opin. Cell Biol. 1995; 7: 301-309Crossref PubMed Scopus (119) Google Scholar): (i) the outer nuclear membrane, which is contiguous with the membranes of the rough endoplasmic reticulum (RER),1 (ii) the pore membranes, which connect outer and inner nuclear membranes and are associated with the nuclear pore complexes, and (iii) the inner nuclear membrane (INM), which is adjacent to the nuclear lamina, a mesh-work of intermediate filament proteins termed lamins (3.Aebi U. Cohn J. Buhle L. Gerace L. Nature. 1986; 323: 560-564Crossref PubMed Scopus (683) Google Scholar). Inner and outer nuclear membranes enclose the perinuclear space that continues into that of the RER. While the outer nuclear membrane shares the membrane proteins of the RER, the INM contains a distinct set of integral proteins (4.Rolls M.M. Stein P.A. Taylor S.S. Ha E. McKeon F. Rapoport T.A. J. Cell Biol. 1999; 146: 29-43Crossref PubMed Scopus (156) Google Scholar), including the lamin B receptor (LBR) (5.Worman H.J. Yuan J. Blobel G. Georgatos S.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8531-8534Crossref PubMed Scopus (298) Google Scholar) and the lamina-associated polypeptides 1 and 2 (LAP1 and LAP2) (6.Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 7.Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (148) Google Scholar). In cells that are virus-infected, the various cellular membranes are decorated with specific viral proteins in addition. In the case of herpesviruses that undergo a characteristic nuclear phase of maturation, viral transmembrane glycoproteins are also translocated from the site of their synthesis into the INM, e.g. the glycoprotein B (gB) homologues of Epstein-Barr virus (8.Gong M. Kieff E. J. Virol. 1990; 64: 1507-1516Crossref PubMed Google Scholar, 9.Qualtiere L.F. Pearson G.R. Int. J. Cancer. 1979; 23: 808-817Crossref PubMed Scopus (38) Google Scholar), herpes simplex virus type 1 (HSV-1) (10.Compton T. Courtney R.J. J. Virol. 1984; 49: 594-597Crossref PubMed Google Scholar), and human cytomegalovirus (HCMV) (11.Radsak K. Brucher K.H. Britt W. Shiou H. Schneider D. Kollert A. Virology. 1990; 177: 515-522Crossref PubMed Scopus (26) Google Scholar). The specific localization in the INM not only suggests a role of these proteins in herpesvirus envelopment at the inner nuclear membrane (12.Roizman B. Sears A.E. Fields Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 2231-2295Google Scholar) but also implies that specific mechanisms mediate their transport from the RER to the INM compartment. It is currently not precisely known how this apparently specific targeting of cellular and viral transmembrane proteins is achieved. For soluble proteins, there is clear evidence that nuclear transport depends on specific sequence motifs, e.g. the nuclear localization signal (NLS) of the SV40 T antigen (13.Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (907) Google Scholar, 14.Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1859) Google Scholar) and the nuclear export signal of HIV Rev protein (15.Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (982) Google Scholar). Such signal sequences have been successfully used fore.g. nuclear targeting of reporter proteins. This observation raised the question of whether comparable signal sequences might exist to target transmembrane proteins to the INM. In previous reports, this aspect was studied for the cellular INM integral proteins LBR and LAP2 as well as the gB homologues of HSV-1 and HCMV. In the case of LBR, two nonoverlapping INM targeting signals have been identified (16.Smith S. Blobel G. J. Cell Biol. 1993; 120: 631-637Crossref PubMed Scopus (115) Google Scholar, 17.Soullam B. Worman H.J. J. Cell Biol. 1993; 120: 1093-1100Crossref PubMed Scopus (136) Google Scholar, 18.Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (179) Google Scholar); i.e. the nucleoplasmic amino-terminal domain as well as the first transmembrane segment appeared to confer nuclear translocation. In the case of LAP2, a type II integral membrane protein, the domain required for targeting to the INM has been restricted to 76 amino-terminal amino acids (19.Furukawa K. Fritze C.E. Gerace L. J. Biol. Chem. 1998; 273: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20.Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). For HSV-1 gB, it has been suggested that an INM localization signal resides in the membrane anchor domain (21.Gilbert R. Ghosh K. Rasile L. Ghosh H.P. J. Virol. 1994; 68: 2272-2285Crossref PubMed Google Scholar, 22.Rasile L. Ghosh K. Raviprakash K. Ghosh H.P. J. Virol. 1993; 67: 4856-4866Crossref PubMed Google Scholar, 23.Raviprakash K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar). Our previous experiments have shown that in the case of HCMV gB, the nucleoplasmic COOH-terminal 51 amino acids are essential for targeting to the INM (24.Bogner E. Anheier B. Offner F. Smuda C. Reschke M. Eickmann M. Radsak K. J. Gen. Virol. 1997; 78: 1647-1651Crossref PubMed Scopus (8) Google Scholar). We then asked if a specific signal within this domain might function in a modular fashion to mediate localization of a reporter protein to the INM and also what are the specific sequence requirements for such a function. Further experiments were performed to answer the question of signal conservation between the gB homologues of different herpesviruses. Experimental evidence is presented in this study that a short sequence element (amino acids 865–906) within the cytoplasmic tail of HCMV gB exhibits the specific potential to localize the type 1 transmembrane protein CD8 used as a reporter into the INM. This signal sequence had no detectable NLS activity for a soluble reporter protein and could not be replaced by the NLS of SV40 T antigen. A comparable element was identified in HSV-1 gB. The sequence sufficient for INM localization of the reporter molecule was further restricted to a minimum of only six amino acids. These findings strongly support the concept of a novel short and modular signal sequence that mediates transport of transmembrane proteins to the INM compartment. Unless otherwise indicated, routine chemicals were purchased from Sigma. Enzymes for DNA cloning were obtained from New England Biolabs (Beverly, MA) or and for cell were from of coding for HCMV gB B. E. Reschke M. A. T. Radsak K. J. Gen. Virol. 1993; PubMed Scopus Google Scholar) and coding for HCMV gB residues S. M. W. Radsak K. J. Gen. Virol. PubMed Scopus Google Scholar) has been The coding for CD8 and coding for the protein were by T. T. M. P.A. Cell. Full Text PDF PubMed Scopus Google Scholar). The SV40 T antigen a protein of and the NLS of the SV40 T antigen was by M. The gB coding for HSV-1 gB was by A. for protein in cells were as To for was performed the for were for at for at and for 1 at a of were by at for were obtained from Unless otherwise indicated, J. T. A Ed. Scholar) were used for DNA DNA and of were by in which was used to coding sequences were also by DNA performed on a model DNA and were For the and was with and the were between these The of an and a site and specific amino acid sequences of HCMV gB for or HSV-1 gB for were and for and for and for and for and for and for and for and for For the and was with and and an the of CD8 with the of for was between these and the was and and the membrane anchor of HCMV gB or HSV-1 gB were The that an and a site were and for and for For the of the the was with and and an the residues of HCMV gB for was between these The of was and To the SV40 T the was with and with the from the was in the and To the and the in was used as by the The was To the gB and was with and these an with the was and The and was used as a For of the and the was with and and the DNA were The was used with the in a with the for for for for for for for cells were in and 2 were at with DNA as by the was at by on cells in of with and by cell fractionation with cells in of were to between and by with monoclonal specific for the or were with of and antibody cells on as well as on were with at for were for with of the HCMV monoclonal antibody L. C. Virology. 1988; PubMed Scopus Google Scholar), HSV-1 K. PubMed Scopus Google Scholar), G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), or by a with in were with a with cells in were with and with of for 1 was with of activity of of for used for cell fractionation of and Cell were with by into and by Cell were in and 1 and in 1 of the at 4 for to the of 1 of K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar). The was with for on an of was were by at for and the was used as the The nuclear was in and and at for a of in The were with and The of the nuclear was by In cell fractionation was by an properties for of nuclear from membrane as by J. D. a Ed. Scholar). In in cells were in of and with of a type of a two of were to to and the was a of in at in a of a were the to the of the membranes at the were in or to a in For of an of the nuclear was on For the nuclear was in nuclear and and were to the of and of the were at for to nuclear and of the were by with protein to at 4 with or were for 2 at 4 protein with six of with of and and with of and the were to gel electrophoresis Nature. PubMed Scopus Google Scholar) and and J. PubMed Scopus Google Scholar) of the experiments were in to that the of cell fractionation and proteins from in the INM (4.Rolls M.M. Stein P.A. Taylor S.S. Ha E. McKeon F. Rapoport T.A. J. Cell Biol. 1999; 146: 29-43Crossref PubMed Scopus (156) Google Scholar, 23.Raviprakash K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar). For this of cells were with HCMV gB B. E. Reschke M. A. T. Radsak K. J. Gen. Virol. 1993; PubMed Scopus Google Scholar) or with an CD8 T. M. P.A. Cell. Full Text PDF PubMed Scopus Google Scholar) 1 were at and to by the of cell fractionation and as The results shown in 1 that HCMV gB was from nuclear as well as was from but not from nuclear B and This observation strongly suggested that the used the Our previous experiments have shown that translocation of HCMV gB to the INM was on the 51 carboxyl-terminal amino acids (24.Bogner E. Anheier B. Offner F. Smuda C. Reschke M. Eickmann M. Radsak K. J. Gen. Virol. 1997; 78: 1647-1651Crossref PubMed Scopus (8) Google Scholar). In to the question of whether the signal within this domain was of of HCMV gB and the membrane protein CD8 as a reporter were 2 of into localization of the proteins was by 2 In to between localization of the in the RER or INM from nuclear and of cells were as For this nuclear were by with which outer nuclear and RER membranes and not the of the INM K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar, M.M. Stein P.A. Taylor S.S. Ha E. McKeon F. Rapoport T.A. J. Cell Biol. 1999; 146: 29-43Crossref PubMed Scopus (156) Google Scholar). Immunofluorescence of the cells with the monoclonal for as in 2 A. For gB characteristic was obtained of the RER and the nuclear envelope 2 which to that in or cells (24.Bogner E. Anheier B. Offner F. Smuda C. Reschke M. Eickmann M. Radsak K. J. Gen. Virol. 1997; 78: 1647-1651Crossref PubMed Scopus (8) Google Scholar). the of HCMV gB in nuclear INM as well as cytoplasmic membranes including RER and membranes 2 These results suggested that a of HCMV gB was in the INM of with CD8 2 on the in cell and 2 CD8 was not in nuclear by of CD8 with the cytoplasmic tail of HCMV gB 2 showed localization in the RER and at the nuclear 2 and demonstrated its in the nuclear 2 results were obtained were of the amino-terminal of CD8 including the transmembrane domain to carboxyl-terminal residues of HCMV gB 2, the CD8 with the cytoplasmic tail of HCMV gB amino acids was not in the INM 2, of the membrane anchor of CD8 by that of HCMV gB 2 in nuclear 2 from the nuclear These suggested that the carboxyl-terminal amino acids of HCMV gB are sufficient to translocate a transmembrane reporter protein to the INM. were performed to between a NLS of soluble proteins and the signal at the of HCMV gB. For this the NLS of SV40 T antigen or amino acids of HCMV gB were to soluble and localization of the proteins was by This in the in the case of the for the gB cytoplasmic comparable with that of was and This observation suggested that the domain of HCMV gB was not a for a NLS for soluble proteins. the of of the carboxyl-terminal domain of HCMV gB by the NLS on was HCMV gB and HCMV gB with a of the carboxyl-terminal domain as a The obtained for this 4 that of HCMV gB with the carboxyl-terminal 4 no of the protein were from nuclear by 4 these experiments it was that the carboxyl-terminal domain of HCMV gB is distinct from the NLS of soluble proteins. In previous it has been suggested that transport of HSV-1 gB to the INM depends on the transmembrane domain (21.Gilbert R. Ghosh K. Rasile L. Ghosh H.P. J. Virol. 1994; 68: 2272-2285Crossref PubMed Google Scholar, 22.Rasile L. Ghosh K. Raviprakash K. Ghosh H.P. J. Virol. 1993; 67: 4856-4866Crossref PubMed Google Scholar, 23.Raviprakash K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar). In to this of HSV-1 gB were to a of the CD8 protein localization of the proteins was by and by of the from of cells HSV-1 gB nuclear as well as RER gB was from nuclear and which was in to of the membrane protein CD8 of the transmembrane domain of CD8 by that of HSV-1 gB the this protein to the INM In the CD8 with the cytoplasmic tail of HSV-1 gB was translocated to the INM the used the cytoplasmic tail but not the transmembrane domain of HSV-1 gB was sufficient to mediate transport to the INM. To further the domain of the HSV-1 gB cytoplasmic tail CD8 were the cytoplasmic tail with various of the HSV-1 gB cytoplasmic tail This revealed to HCMV gB, the signal of HSV-1 gB not in the of the molecule but within the of the cytoplasmic tail and To identify of the transport signals of HCMV and HSV-1 gB, amino acid sequences of the domains in of the HCMV and of the HSV-1 gB were the shown in revealed only between the two domains the in the case of HCMV gB a was for a segment including a residue in which is to a For HSV-1 gB, on the was for a segment from to For further of the HCMV gB residues HCMV HSV-1 HCMV gB HSV-1 gB HSV-1 HCMV gB HSV-1 with were In to the role of the residues and were in the molecule as well as in the gB of the proteins was by and and as and HCMV gB as well as CD8 as a and These specific of residues and transport of HCMV gB as well as that of the gB to the INM B and and This observation suggested a role of amino acid residues and or at a for translocation of HCMV gB to the INM. experiments to whether the short amino acid of HCMV gB was sufficient to localize a reporter protein to the INM. The amino acid sequence DRLRHR was to CD8 and localization of the protein was by was obtained for the but not for CD8 This was by of This was used in of proteins sufficient To further this fractionation of cells with the in was by a by J. D. a Ed. Scholar) that for of were to with or to with specific specific as the of the nuclear envelope including the outer nuclear membrane, which is contiguous with the which the outer nuclear membrane specific signals were in the case of or but in with the coding for HCMV gB or This observation that the were also in the inner of the nuclear These findings strongly the concept of a novel short and modular signal sequence that mediates transport to the INM. was performed to further the of amino acid residues within the novel signal For this residue of the sequence element was substituted by in the of of the proteins by the of residues and of HCMV gB not the of nuclear was and to that by the the of residues and perinuclear of the The with a of residue membrane and in nuclear was of was by of These that arginine residues in positions 4 and 6 of the sequence DRLRHR are essential for its function as an INM transport The results presented that the amino acid sequence DRLRHR of HCMV gB 885–890) was sufficient to mediate transport of a reporter type transmembrane protein to the INM. A comparable signal was identified in the cytoplasmic tail of HSV-1 gB. residues in positions 4 and 6 of the HCMV gB signal sequence appeared to be essential for its function as a transport Our experiments also that the transport for transmembrane proteins is distinct from the for nuclear of soluble proteins. We to this novel signal sequence as signal for inner nuclear localization of the proteins was by of was as for INM the by to be by cell fractionation K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar, Blobel G. Cell. 1986; Full Text PDF PubMed Scopus Google Scholar) in to between outer and inner nuclear membranes to In the fractionation was used to of outer nuclear membrane (4.Rolls M.M. Stein P.A. Taylor S.S. Ha E. McKeon F. Rapoport T.A. J. Cell Biol. 1999; 146: 29-43Crossref PubMed Scopus (156) Google Scholar, 23.Raviprakash K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar) to the specific these the membrane protein CD8 as well as CD8 the signal of the transmembrane protein were not associated with nuclear These showed that nuclear were not by of cytoplasmic membranes and that the fractionation used were Further of the localization of the proteins by to antibody to HSV-1 gB, findings are in with previous that the transmembrane domain is an essential transport signal for INM translocation (21.Gilbert R. Ghosh K. Rasile L. Ghosh H.P. J. Virol. 1994; 68: 2272-2285Crossref PubMed Google Scholar, 22.Rasile L. Ghosh K. Raviprakash K. Ghosh H.P. J. Virol. 1993; 67: 4856-4866Crossref PubMed Google Scholar, 23.Raviprakash K. Rasile L. Ghosh K. Ghosh H.P. J. Biol. Chem. 1990; 265: 1777-1782Abstract Full Text PDF PubMed Google Scholar). This the different by these used virus protein as a A with this is that virus is known to be into the INM J. Cell Biol. PubMed Scopus Google Scholar). on that amino acid residues in the carboxyl-terminal of HSV-1 gB are the of its transport to the INM. The of the nuclear translocation within the cytoplasmic of gB of HCMV and in their Inner nuclear membrane targeting has been studied for LBR and In the case of LBR, the amino-terminal domain as well as the first transmembrane segment of LBR were to mediate nuclear localization (16.Smith S. Blobel G. J. Cell Biol. 1993; 120: 631-637Crossref PubMed Scopus (115) Google Scholar, 17.Soullam B. Worman H.J. J. Cell Biol. 1993; 120: 1093-1100Crossref PubMed Scopus (136) Google Scholar, 18.Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (179) Google Scholar). the a signal comparable with that for HCMV gB was identified in the nucleoplasmic tail of LBR of the LBR It be of in this context to whether this LBR signal is sufficient for reporter protein For LAP2, which is also a type II transmembrane protein, translocation to the INM has been shown to be by amino acid residues in the nucleoplasmic amino of the molecule (19.Furukawa K. Fritze C.E. Gerace L. J. Biol. Chem. 1998; 273: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20.Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). not to the HCMV gB signal In be to identify signal The results of the study that a different from sufficient to target soluble proteins into the is to target a membrane protein to the INM. This strongly suggests that the two protein transmembrane and soluble proteins, are to the nuclear distinct It be that HCMV gB is to the INM by the membranes of the nuclear membrane and the nuclear This was for transport of the LBR (16.Smith S. Blobel G. J. Cell Biol. 1993; 120: 631-637Crossref PubMed Scopus (115) Google Scholar, 17.Soullam B. Worman H.J. J. Cell Biol. 1993; 120: 1093-1100Crossref PubMed Scopus (136) Google Scholar, 18.Soullam B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (179) Google Scholar, J. Smith Worman H.J. J. J. Cell Biol. 1997; PubMed Scopus Google Scholar), the LAP2 (19.Furukawa K. Fritze C.E. Gerace L. J. Biol. Chem. 1998; 273: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20.Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar), and the pore membrane protein Blobel G. J. Cell Biol. PubMed Scopus Google Scholar). The pore could a have that exist at the of the pore J. Biol. 1995; PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). INM proteins specific have shown that of the amino-terminal tail of the LBR of the protein in the INM B. Worman H.J. J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (179) Google Scholar). This observation is with the Such could be by of proteins at the INM by with nuclear proteins. The that LBR, and LAP2 with lamins A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (148) Google Scholar, Worman H.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) suggests that lamin in function to proteins in the INM. the nuclear envelope targeting domain of LAP2 to be with its lamin (19.Furukawa K. Fritze C.E. Gerace L. J. Biol. Chem. 1998; 273: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). are in to this for HCMV gB. the novel signal sequence of HCMV gB be as an to identify a distinct for translocation of transmembrane proteins to the INM. We are to T. M. A. and B. Britt of for DNA and We also G. for and of the and S. for We are to E. M. M. W. D. K. S. and T. for