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• W2022711087 abstract "The hepatitis C virus (HCV) NS2 protein is a hydrophobic protein. Previous studies indicate that this protein is an integral membrane protein, which is targeted to the membrane of the endoplasmic reticulum (ER) by the signal sequence located in its preceding p7 protein. In this report, we demonstrate that the membrane association of NS2 is p7-independent and occurs co-translationally. Further deletion-mapping studies suggest the presence of two internal signal sequences in NS2. These two internal signal sequences, which are located within amino acids 839–883 and amino acids 928–960, could target the α-globin reporter, a cytosolic protein, to the membrane compartments in HuH7 hepatoma cells. The presence of multiple signal sequences for its membrane association suggests that NS2 has multiple transmembrane domains. The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen. Based on these results, a model for the NS2 membrane topology is presented. The hepatitis C virus (HCV) NS2 protein is a hydrophobic protein. Previous studies indicate that this protein is an integral membrane protein, which is targeted to the membrane of the endoplasmic reticulum (ER) by the signal sequence located in its preceding p7 protein. In this report, we demonstrate that the membrane association of NS2 is p7-independent and occurs co-translationally. Further deletion-mapping studies suggest the presence of two internal signal sequences in NS2. These two internal signal sequences, which are located within amino acids 839–883 and amino acids 928–960, could target the α-globin reporter, a cytosolic protein, to the membrane compartments in HuH7 hepatoma cells. The presence of multiple signal sequences for its membrane association suggests that NS2 has multiple transmembrane domains. The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen. Based on these results, a model for the NS2 membrane topology is presented. hepatitis C virus endoplasmic reticulum HCV E1 envelope protein HCV E2 envelope protein nucleotide(s) cytomegalovirus immediate early amino acid(s) microsomal membranes Tris-buffered saline endoglycosidase H radioimmune precipitation Hepatitis C virus (HCV)1is an important human pathogen that can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma (1Colombo M. Kuo G. Choo Q.L. Donato M.F. Del Ninno E. Tommasini M.A. Dioguardi N. Houghton M. Lancet. 1989; 2: 1006-1008Abstract PubMed Scopus (607) Google Scholar, 2Di Bisceglie A.M. Order S.E. Klein J.L. Waggoner J.G. Sjogren M.H. Kuo G. Houghton M. Choo Q.L. Hoofnagle J.H. Am. J. Gastroenterol. 1991; 86: 335-338PubMed Google Scholar, 3Kiyosawa K. Sodeyama T. Tanaka E. Gibo Y. Yoshizawa K. Nakano Y. Furuta S. Akahane Y. Nishioka K. Purcell R.H. Alter H.J. Hepatology. 1990; 12: 671-675Crossref PubMed Scopus (1181) Google Scholar, 4Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. et al.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1079) Google Scholar). This virus has a 9.6-kb positive-stranded RNA genome that codes for a polyprotein with a length of slightly more than 3000 amino acids. The HCV structural proteins, core, E1, and E2, are located at the amino terminus and are released from the polyprotein by the cellular signal peptidase located in the lumen of the endoplasmic reticulum (ER) (5Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (579) Google Scholar, 6Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 7Tomei L. Failla C. Santolini E., De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar). The non-structural proteins, NS3, NS4A, NS4B, NS5A, and NS5B, are located at the carboxyl terminus and are separated from each other by the protease activity residing within the NS3 protein (7Tomei L. Failla C. Santolini E., De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar, 8D'Souza E.D. O'Sullivan E. Amphlett E.M. Rowlands D.J. Sangar D.V. Clarke B.E. J. Gen. Virol. 1994; 75: 3469-3476Crossref PubMed Scopus (39) Google Scholar, 9Hijikata M. Mizushima H. Akagi T. Mori S. Kakiuchi N. Kato N. Tanaka T. Kimura K. Shimotohno K. J. Virol. 1993; 67: 4665-4675Crossref PubMed Google Scholar).Between E2 and NS3 there are two additional proteins named p7 and NS2. p7 may or may not be separated from the E2 protein (10Lin C. Lindenbach B.D. Pragai B.M. McCourt D.W. Rice C.M. J. Virol. 1994; 68: 5063-5073Crossref PubMed Google Scholar) and has been shown to contain a signal sequence to direct the downstream NS2 protein to the membranes (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar). NS2 is separated from its preceding p7 protein by the signal peptidase (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar). The function of NS2 in the HCV life cycle is unclear. Its deletion did not abolish the replication of HCV RNA in cell cultures, indicating that it is not required for viral RNA replication (12Blight K.J. Kolykhalov A.A. Rice C.M. Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1271) Google Scholar, 13Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2480) Google Scholar). The separation of NS2 and its following NS3 protein requires most of the NS2 sequence and its adjacent NS3 protease domain (14Pieroni L. Santolini E. Fipaldini C. Pacini L. Migliaccio G. La Monica N. J. Virol. 1997; 71: 6373-6380Crossref PubMed Google Scholar).The deduced amino acid sequence of NS2 indicates that it is a hydrophobic protein. Previous studies indicate that NS2 is a non-glycosylated integral membrane protein and mostly not exposed in the cytosol (15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). However, the membrane topology of this protein remains unclear. In this report, we have studied the molecular mechanism that regulates the membrane translocation of NS2. Interestingly, our results indicate that the membrane-association of NS2 is p7-independent. Further studies indicate that NS2 contains at least two internal signal sequences for its membrane association and likely has multiple transmembrane domains.DISCUSSIONPrevious studies indicate that the HCV NS2 protein is an integral membrane protein that is targeted to the ER membrane by a signal sequence located in its preceding p7 protein (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar, 15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). In this report, we have further studied how NS2 associates with the membranes. In agreement with the earlier studies, we found that the NS2 protein was separated from the preceding p7 sequence only in the presence of membranes (Fig. 1A), indicative of separation by the signal peptidase. Interestingly, we also found that NS2, in the absence of p7, could become membrane-associated (Fig. 1B). This membrane association is supported by the immunofluorescence double-staining experiment using the ER-associated calreticulin as the ER marker (Fig.2B). Because this membrane association could not be abolished by treating the membranes with 1 m NaCl or by extraction with the chaotropic alkaline carbonate buffer (Fig. 4), NS2 was apparently an integral membrane protein. The association of NS2 with the membranes only occurred co-translationally (Fig. 3), suggesting the presence of internal signal sequences in NS2. This possibility was tested by the deletion-mapping experiments, which revealed two internal signal sequences located within aa 839–883 and aa 928–960 (Figs. Figure 5, Figure 6, Figure 7). These two sequences could target the α-globin reporter, a cytosolic protein, to the membranes in HuH7 cells (Figs. 7 and 8).The identification of the two internal signal sequences suggests that NS2 is a type III integral membrane protein with multiple transmembrane domains (20Matlack K.E. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Singer S.J. Annu. Rev. Cell Biol. 1990; 6: 247-296Crossref PubMed Scopus (239) Google Scholar). To further study the membrane topology of NS2, we performed the glycosylation studies by introducing glycosylation sites into various regions of the NS2 sequence. Interestingly, the glycosylation site introduced near the amino terminus of NS2 was used independent of the p7 sequence (Fig. 9A). These results indicate that the amino terminus of NS2 can be localized to the ER lumen independent of p7. Because the presence of p7 did not significantly increase the glycosylation efficiency of NS2–812G (Fig.9A), the role of the p7 signal sequence in the membrane translocation of NS2 is unclear. The p7 signal sequence may be needed only for the separation of NS2 from its preceding HCV sequences by signal peptidases.The hydrophobicity plot of NS2 based on its deduced amino acid sequence is shown in Fig. 10A. The sequence aa 814–835 near the amino terminus is highly hydrophobic. It is conceivable that this hydrophobic domain serves as the first transmembrane domain that will have a type I topology, because the amino terminus of NS2 is localized in the ER lumen. If this is correct, the hydrophilic sequence immediately following aa 835 is expected to be localized in the cytosol. The observation that the glycosylation site introduced at aa 839 was not used is consistent with this speculation (Fig. 10B). The first internal signal sequence was mapped between aa 839 and 883 (Figs. Figure 5, Figure 6, Figure 7). This sequence likely contains the second transmembrane domain. As the glycosylation site introduced at aa 869 was used for glycosylation, the second transmembrane domain may be residing between aa 843 and 866, a sequence that is largely hydrophobic (Fig. 10A). This second transmembrane domain will have a type II topology with the amino terminus in the cytosol and the carboxyl terminus in the ER lumen.The sequence from aa 872 to 919 is highly hydrophobic (Fig.10A). This sequence may contain a third transmembrane domain. If this is correct, this transmembrane domain is expected to have a type I topology with the amino terminus in the ER lumen and the carboxyl terminus in the cytosol. The second signal sequence was identified between aa 928 and 960. This sequence likely contains the fourth transmembrane domain. The sequence from aa 928 to 956 is highly hydrophobic and may serve this function (Fig. 10A). If this second signal sequence indeed contains another transmembrane domain, it will likely have a type II topology with its carboxyl terminus localized to the ER lumen, because the glycosylation site introduced at aa 988 was used for glycosylation (Fig. 9B, also see Ref.15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). The carboxyl terminus of NS2 is predicted to localize in the ER lumen, because the glycosylation site introduced at aa 1018 near the carboxyl terminus was used for glycosylation (Fig. 9B). This result is consistent with the glycosylation studies of Santoliniet al. (15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar), who also predicted that the carboxyl terminus of NS2 would be localized to the ER lumen. A model of the NS2 topology in the membrane is illustrated in Fig. 10B. This model predicts that the amino terminus and the carboxyl terminus are localized to the ER lumen and that NS2 has a total of four alternating type I and type II transmembrane domains.In conclusion, our studies have allowed us to identify two internal signal sequences in the NS2 sequence, which allowed us to propose a model that NS2 has a type III membrane topology with multiple transmembrane domains. This model will now enable us to further explore the possible functions of NS2, which have remained largely elusive. Hepatitis C virus (HCV)1is an important human pathogen that can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma (1Colombo M. Kuo G. Choo Q.L. Donato M.F. Del Ninno E. Tommasini M.A. Dioguardi N. Houghton M. Lancet. 1989; 2: 1006-1008Abstract PubMed Scopus (607) Google Scholar, 2Di Bisceglie A.M. Order S.E. Klein J.L. Waggoner J.G. Sjogren M.H. Kuo G. Houghton M. Choo Q.L. Hoofnagle J.H. Am. J. Gastroenterol. 1991; 86: 335-338PubMed Google Scholar, 3Kiyosawa K. Sodeyama T. Tanaka E. Gibo Y. Yoshizawa K. Nakano Y. Furuta S. Akahane Y. Nishioka K. Purcell R.H. Alter H.J. Hepatology. 1990; 12: 671-675Crossref PubMed Scopus (1181) Google Scholar, 4Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. et al.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1079) Google Scholar). This virus has a 9.6-kb positive-stranded RNA genome that codes for a polyprotein with a length of slightly more than 3000 amino acids. The HCV structural proteins, core, E1, and E2, are located at the amino terminus and are released from the polyprotein by the cellular signal peptidase located in the lumen of the endoplasmic reticulum (ER) (5Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (579) Google Scholar, 6Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 7Tomei L. Failla C. Santolini E., De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar). The non-structural proteins, NS3, NS4A, NS4B, NS5A, and NS5B, are located at the carboxyl terminus and are separated from each other by the protease activity residing within the NS3 protein (7Tomei L. Failla C. Santolini E., De Francesco R. La Monica N. J. Virol. 1993; 67: 4017-4026Crossref PubMed Google Scholar, 8D'Souza E.D. O'Sullivan E. Amphlett E.M. Rowlands D.J. Sangar D.V. Clarke B.E. J. Gen. Virol. 1994; 75: 3469-3476Crossref PubMed Scopus (39) Google Scholar, 9Hijikata M. Mizushima H. Akagi T. Mori S. Kakiuchi N. Kato N. Tanaka T. Kimura K. Shimotohno K. J. Virol. 1993; 67: 4665-4675Crossref PubMed Google Scholar). Between E2 and NS3 there are two additional proteins named p7 and NS2. p7 may or may not be separated from the E2 protein (10Lin C. Lindenbach B.D. Pragai B.M. McCourt D.W. Rice C.M. J. Virol. 1994; 68: 5063-5073Crossref PubMed Google Scholar) and has been shown to contain a signal sequence to direct the downstream NS2 protein to the membranes (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar). NS2 is separated from its preceding p7 protein by the signal peptidase (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar). The function of NS2 in the HCV life cycle is unclear. Its deletion did not abolish the replication of HCV RNA in cell cultures, indicating that it is not required for viral RNA replication (12Blight K.J. Kolykhalov A.A. Rice C.M. Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1271) Google Scholar, 13Lohmann V. Korner F. Koch J. Herian U. Theilmann L. Bartenschlager R. Science. 1999; 285: 110-113Crossref PubMed Scopus (2480) Google Scholar). The separation of NS2 and its following NS3 protein requires most of the NS2 sequence and its adjacent NS3 protease domain (14Pieroni L. Santolini E. Fipaldini C. Pacini L. Migliaccio G. La Monica N. J. Virol. 1997; 71: 6373-6380Crossref PubMed Google Scholar). The deduced amino acid sequence of NS2 indicates that it is a hydrophobic protein. Previous studies indicate that NS2 is a non-glycosylated integral membrane protein and mostly not exposed in the cytosol (15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). However, the membrane topology of this protein remains unclear. In this report, we have studied the molecular mechanism that regulates the membrane translocation of NS2. Interestingly, our results indicate that the membrane-association of NS2 is p7-independent. Further studies indicate that NS2 contains at least two internal signal sequences for its membrane association and likely has multiple transmembrane domains. DISCUSSIONPrevious studies indicate that the HCV NS2 protein is an integral membrane protein that is targeted to the ER membrane by a signal sequence located in its preceding p7 protein (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar, 15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). In this report, we have further studied how NS2 associates with the membranes. In agreement with the earlier studies, we found that the NS2 protein was separated from the preceding p7 sequence only in the presence of membranes (Fig. 1A), indicative of separation by the signal peptidase. Interestingly, we also found that NS2, in the absence of p7, could become membrane-associated (Fig. 1B). This membrane association is supported by the immunofluorescence double-staining experiment using the ER-associated calreticulin as the ER marker (Fig.2B). Because this membrane association could not be abolished by treating the membranes with 1 m NaCl or by extraction with the chaotropic alkaline carbonate buffer (Fig. 4), NS2 was apparently an integral membrane protein. The association of NS2 with the membranes only occurred co-translationally (Fig. 3), suggesting the presence of internal signal sequences in NS2. This possibility was tested by the deletion-mapping experiments, which revealed two internal signal sequences located within aa 839–883 and aa 928–960 (Figs. Figure 5, Figure 6, Figure 7). These two sequences could target the α-globin reporter, a cytosolic protein, to the membranes in HuH7 cells (Figs. 7 and 8).The identification of the two internal signal sequences suggests that NS2 is a type III integral membrane protein with multiple transmembrane domains (20Matlack K.E. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Singer S.J. Annu. Rev. Cell Biol. 1990; 6: 247-296Crossref PubMed Scopus (239) Google Scholar). To further study the membrane topology of NS2, we performed the glycosylation studies by introducing glycosylation sites into various regions of the NS2 sequence. Interestingly, the glycosylation site introduced near the amino terminus of NS2 was used independent of the p7 sequence (Fig. 9A). These results indicate that the amino terminus of NS2 can be localized to the ER lumen independent of p7. Because the presence of p7 did not significantly increase the glycosylation efficiency of NS2–812G (Fig.9A), the role of the p7 signal sequence in the membrane translocation of NS2 is unclear. The p7 signal sequence may be needed only for the separation of NS2 from its preceding HCV sequences by signal peptidases.The hydrophobicity plot of NS2 based on its deduced amino acid sequence is shown in Fig. 10A. The sequence aa 814–835 near the amino terminus is highly hydrophobic. It is conceivable that this hydrophobic domain serves as the first transmembrane domain that will have a type I topology, because the amino terminus of NS2 is localized in the ER lumen. If this is correct, the hydrophilic sequence immediately following aa 835 is expected to be localized in the cytosol. The observation that the glycosylation site introduced at aa 839 was not used is consistent with this speculation (Fig. 10B). The first internal signal sequence was mapped between aa 839 and 883 (Figs. Figure 5, Figure 6, Figure 7). This sequence likely contains the second transmembrane domain. As the glycosylation site introduced at aa 869 was used for glycosylation, the second transmembrane domain may be residing between aa 843 and 866, a sequence that is largely hydrophobic (Fig. 10A). This second transmembrane domain will have a type II topology with the amino terminus in the cytosol and the carboxyl terminus in the ER lumen.The sequence from aa 872 to 919 is highly hydrophobic (Fig.10A). This sequence may contain a third transmembrane domain. If this is correct, this transmembrane domain is expected to have a type I topology with the amino terminus in the ER lumen and the carboxyl terminus in the cytosol. The second signal sequence was identified between aa 928 and 960. This sequence likely contains the fourth transmembrane domain. The sequence from aa 928 to 956 is highly hydrophobic and may serve this function (Fig. 10A). If this second signal sequence indeed contains another transmembrane domain, it will likely have a type II topology with its carboxyl terminus localized to the ER lumen, because the glycosylation site introduced at aa 988 was used for glycosylation (Fig. 9B, also see Ref.15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). The carboxyl terminus of NS2 is predicted to localize in the ER lumen, because the glycosylation site introduced at aa 1018 near the carboxyl terminus was used for glycosylation (Fig. 9B). This result is consistent with the glycosylation studies of Santoliniet al. (15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar), who also predicted that the carboxyl terminus of NS2 would be localized to the ER lumen. A model of the NS2 topology in the membrane is illustrated in Fig. 10B. This model predicts that the amino terminus and the carboxyl terminus are localized to the ER lumen and that NS2 has a total of four alternating type I and type II transmembrane domains.In conclusion, our studies have allowed us to identify two internal signal sequences in the NS2 sequence, which allowed us to propose a model that NS2 has a type III membrane topology with multiple transmembrane domains. This model will now enable us to further explore the possible functions of NS2, which have remained largely elusive. Previous studies indicate that the HCV NS2 protein is an integral membrane protein that is targeted to the ER membrane by a signal sequence located in its preceding p7 protein (11Mizushima H. Hijikata M. Tanji Y. Kimura K. Shimotohno K. J. Virol. 1994; 68: 2731-2734Crossref PubMed Google Scholar, 15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). In this report, we have further studied how NS2 associates with the membranes. In agreement with the earlier studies, we found that the NS2 protein was separated from the preceding p7 sequence only in the presence of membranes (Fig. 1A), indicative of separation by the signal peptidase. Interestingly, we also found that NS2, in the absence of p7, could become membrane-associated (Fig. 1B). This membrane association is supported by the immunofluorescence double-staining experiment using the ER-associated calreticulin as the ER marker (Fig.2B). Because this membrane association could not be abolished by treating the membranes with 1 m NaCl or by extraction with the chaotropic alkaline carbonate buffer (Fig. 4), NS2 was apparently an integral membrane protein. The association of NS2 with the membranes only occurred co-translationally (Fig. 3), suggesting the presence of internal signal sequences in NS2. This possibility was tested by the deletion-mapping experiments, which revealed two internal signal sequences located within aa 839–883 and aa 928–960 (Figs. Figure 5, Figure 6, Figure 7). These two sequences could target the α-globin reporter, a cytosolic protein, to the membranes in HuH7 cells (Figs. 7 and 8). The identification of the two internal signal sequences suggests that NS2 is a type III integral membrane protein with multiple transmembrane domains (20Matlack K.E. Mothes W. Rapoport T.A. Cell. 1998; 92: 381-390Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 21Singer S.J. Annu. Rev. Cell Biol. 1990; 6: 247-296Crossref PubMed Scopus (239) Google Scholar). To further study the membrane topology of NS2, we performed the glycosylation studies by introducing glycosylation sites into various regions of the NS2 sequence. Interestingly, the glycosylation site introduced near the amino terminus of NS2 was used independent of the p7 sequence (Fig. 9A). These results indicate that the amino terminus of NS2 can be localized to the ER lumen independent of p7. Because the presence of p7 did not significantly increase the glycosylation efficiency of NS2–812G (Fig.9A), the role of the p7 signal sequence in the membrane translocation of NS2 is unclear. The p7 signal sequence may be needed only for the separation of NS2 from its preceding HCV sequences by signal peptidases. The hydrophobicity plot of NS2 based on its deduced amino acid sequence is shown in Fig. 10A. The sequence aa 814–835 near the amino terminus is highly hydrophobic. It is conceivable that this hydrophobic domain serves as the first transmembrane domain that will have a type I topology, because the amino terminus of NS2 is localized in the ER lumen. If this is correct, the hydrophilic sequence immediately following aa 835 is expected to be localized in the cytosol. The observation that the glycosylation site introduced at aa 839 was not used is consistent with this speculation (Fig. 10B). The first internal signal sequence was mapped between aa 839 and 883 (Figs. Figure 5, Figure 6, Figure 7). This sequence likely contains the second transmembrane domain. As the glycosylation site introduced at aa 869 was used for glycosylation, the second transmembrane domain may be residing between aa 843 and 866, a sequence that is largely hydrophobic (Fig. 10A). This second transmembrane domain will have a type II topology with the amino terminus in the cytosol and the carboxyl terminus in the ER lumen. The sequence from aa 872 to 919 is highly hydrophobic (Fig.10A). This sequence may contain a third transmembrane domain. If this is correct, this transmembrane domain is expected to have a type I topology with the amino terminus in the ER lumen and the carboxyl terminus in the cytosol. The second signal sequence was identified between aa 928 and 960. This sequence likely contains the fourth transmembrane domain. The sequence from aa 928 to 956 is highly hydrophobic and may serve this function (Fig. 10A). If this second signal sequence indeed contains another transmembrane domain, it will likely have a type II topology with its carboxyl terminus localized to the ER lumen, because the glycosylation site introduced at aa 988 was used for glycosylation (Fig. 9B, also see Ref.15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar). The carboxyl terminus of NS2 is predicted to localize in the ER lumen, because the glycosylation site introduced at aa 1018 near the carboxyl terminus was used for glycosylation (Fig. 9B). This result is consistent with the glycosylation studies of Santoliniet al. (15Santolini E. Pacini L. Fipaldini C. Migliaccio G. Monica N. J. Virol. 1995; 69: 7461-7471Crossref PubMed Google Scholar), who also predicted that the carboxyl terminus of NS2 would be localized to the ER lumen. A model of the NS2 topology in the membrane is illustrated in Fig. 10B. This model predicts that the amino terminus and the carboxyl terminus are localized to the ER lumen and that NS2 has a total of four alternating type I and type II transmembrane domains. In conclusion, our studies have allowed us to identify two internal signal sequences in the NS2 sequence, which allowed us to propose a model that NS2 has a type III membrane topology with multiple transmembrane domains. This model will now enable us to further explore the possible functions of NS2, which have remained largely elusive. We thank Stephanie Tang for her participation in the early phase of this research project and Drs. Ari Bergwerk and Jinah Choi for the critical reading of the manuscript." @default.
• W2022711087 created "2016-06-24" @default.
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• W2022711087 date "2002-09-01" @default.
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• W2022711087 title "Membrane Topology of the Hepatitis C Virus NS2 Protein" @default.
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