Enhanced α1 Microglobulin Secretion from Hepatitis E Virus ORF3-expressing Human Hepatoma Cells Is Mediated by the Tumor Susceptibility Gene 101

Viruses are known to exploit the host cell machinery for their benefit during different stages of their life cycle within the infected host. One of the major challenges for a virus during the early stages of infection is to escape recognition by the host immune system. Viruses have adopted many novel strategies to evade the host immune response or to create an immune suppressed environment. An earlier study in our laboratory has demonstrated that the ORF3 protein of the hepatitis E virus expedites the secretion of α1 microglobulin, an immunosuppressant molecule. Based on this observation, we proposed that enhanced secretion of α1 microglobulin may help maintain an immunosuppressed milieu around the infected hepatocyte (Tyagi, S., Surjit, M., Roy, A. K., Jameel, S., and Lal, S. K. (2004) J. Biol. Chem. 279, 29308–29319). In the present study, we discovered that the ability of the ORF3 protein to expedite α1 microglobulin secretion is attributed to the PSAP motif present at the C terminus of the former. The ORF3 protein was able to associate with the tumor susceptibility gene 101 (TSG101) through the PSAP motif. Further, a PSAP motif-mutated ORF3 protein was unable to associate with TSG101 and also lost its ability to enhance the secretion of α1 microglobulin. In addition, the ORF3 protein was found to associate simultaneously with TSG101 and α1 microglobulin because all three of them were co-precipitated as a ternary complex. Finally, a dominant negative mutant of the VPS4 protein was shown to block the enhanced α1 microglobulin secretion in ORF3-expressing hepatocytes. These results suggest a mechanism by which the ORF3 protein exploits the endosomal sorting machinery to enhance the secretion of an immunosuppressant molecule (α1 microglobulin) from the cultured hepatocytes. Viruses are known to exploit the host cell machinery for their benefit during different stages of their life cycle within the infected host. One of the major challenges for a virus during the early stages of infection is to escape recognition by the host immune system. Viruses have adopted many novel strategies to evade the host immune response or to create an immune suppressed environment. An earlier study in our laboratory has demonstrated that the ORF3 protein of the hepatitis E virus expedites the secretion of α1 microglobulin, an immunosuppressant molecule. Based on this observation, we proposed that enhanced secretion of α1 microglobulin may help maintain an immunosuppressed milieu around the infected hepatocyte (Tyagi, S., Surjit, M., Roy, A. K., Jameel, S., and Lal, S. K. (2004) J. Biol. Chem. 279, 29308–29319). In the present study, we discovered that the ability of the ORF3 protein to expedite α1 microglobulin secretion is attributed to the PSAP motif present at the C terminus of the former. The ORF3 protein was able to associate with the tumor susceptibility gene 101 (TSG101) through the PSAP motif. Further, a PSAP motif-mutated ORF3 protein was unable to associate with TSG101 and also lost its ability to enhance the secretion of α1 microglobulin. In addition, the ORF3 protein was found to associate simultaneously with TSG101 and α1 microglobulin because all three of them were co-precipitated as a ternary complex. Finally, a dominant negative mutant of the VPS4 protein was shown to block the enhanced α1 microglobulin secretion in ORF3-expressing hepatocytes. These results suggest a mechanism by which the ORF3 protein exploits the endosomal sorting machinery to enhance the secretion of an immunosuppressant molecule (α1 microglobulin) from the cultured hepatocytes. The endosomal protein sorting pathway is a highly dynamic system in cells. It coordinates protein transport from both the biosynthetic and endocytic pathways and hence plays an important role in sorting proteins destined to be degraded, recycled, or secreted. The machinery responsible for this tightly regulated protein sorting process in the endosomal pathway consists of a series of proteins including three protein complexes: endosomal sorting complex required for transport (ESCRT) 4The abbreviations used are: ESCRT, endosomal sorting complex required for transport; AD, activation domain; BD, binding domain; 3-AT, 3-amino-1,2,4-triazole. TSG101, tumor susceptibility gene 101; α1m, α1 microglobulin; HEV, hepatitis E virus; HIV-1, human immunodeficiency virus, type 1; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; FITC, fluorescein isothiocyanate; SD, synthetic dextrose complete medium. 4The abbreviations used are: ESCRT, endosomal sorting complex required for transport; AD, activation domain; BD, binding domain; 3-AT, 3-amino-1,2,4-triazole. TSG101, tumor susceptibility gene 101; α1m, α1 microglobulin; HEV, hepatitis E virus; HIV-1, human immunodeficiency virus, type 1; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; FITC, fluorescein isothiocyanate; SD, synthetic dextrose complete medium. I, II, and III (1Raiborg C. Rusten T.E. Stenmark H. Curr. Opin. Cell Biol. 2003; 15: 446-455Crossref PubMed Scopus (405) Google Scholar). In recent years, several RNA viruses have been demonstrated to reprogram the host endosomal protein sorting machinery to their benefit (2Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). For example, viruses such as HIV-1 and Ebola do not encode their own machineries for budding. Instead they bear two proline-rich motifs in their late domain: P(S/T)AP and PPXY (where X is any amino acid), which have been proved to be essential for viral budding (3Licata J.M. Simpson-Holley M. Wright N.T. Han Z. Paragas J. Harty R.N. J. Virol. 2003; 77: 1812-1819Crossref PubMed Scopus (231) Google Scholar, 4Goff A. Ehrlich L.S. Cohen S.N. Carter C.A. J. Virol. 2003; 77: 9173-9182Crossref PubMed Scopus (80) Google Scholar). The P(S/T)AP motif of the HIV-1 Gag late domain has been shown to bind to TSG101 in a manner analogous to that of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and hijacks the ESCRT complex for virus budding (5Amara A. Littman D.R. J. Cell Biol. 2003; 162: 371-375Crossref PubMed Scopus (32) Google Scholar). Similarly, the VP40 gene product of Ebola virus binds TSG101 via the PSAP motif (6Timmins J. Schoehn G. Ricard-Blum S. Scianimanico S. Vernet T. Ruigrok R.W. Weissenhorn W. J. Mol. Biol. 2003; 326: 493-502Crossref PubMed Scopus (170) Google Scholar) for virus budding. TSG101 is a central mediator of the endosomal trafficking process. It associates with HRS through the PSAP motif (HRS recognizes ubiquitinated cargo in normal cellular context). This association enables the cargo to be targeted to the late endosomes and subsequently get sorted into lysosomes or recycled back. Therefore, TSG101 has been an attractive target for viruses to exploit the host endosomal trafficking machinery to bud off the host cell. Like P(S/T)AP, the PPXY motif present in the late domain of capsid proteins has also been shown to be important for viral budding. This motif is a preferred binding motif for WW domains found in the Nedd4 family of HECT domain-containing ubiquitin ligases (7Harty R.N. Brown M.E. Wang G. Huibregtse J. Hayes F.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13871-13876Crossref PubMed Scopus (374) Google Scholar). Both HIV-1 Gag and the VP40 protein of Ebola virus bind Nedd4 and get ubiquitinated, which aids in recognition by HRS/TSG101 and virus budding (3Licata J.M. Simpson-Holley M. Wright N.T. Han Z. Paragas J. Harty R.N. J. Virol. 2003; 77: 1812-1819Crossref PubMed Scopus (231) Google Scholar, 8Pornillos O. Alam S.L. Rich R.L. Myszka D.G. Davis D.R. Sundquist W.I. EMBO J. 2002; 21: 2397-2406Crossref PubMed Scopus (242) Google Scholar). Another crucial function for which viruses hijack the endosomal sorting machinery is to subvert the immune surveillance process. An example of this phenomenon is provided by the k3 gene product of Kaposi sarcoma-associated Herpesvirus. The k3 protein is an ubiquitin ligase that ubiquitinates newly synthesized major histocompatibility complex class I molecules, thus targeting them for lysosomal degradation in a TSG101-dependent manner (10Hewitt E.W. Duncan L. Mufti D. Baker J. Stevenson P.G. Lehner P.J. EMBO J. 2002; 21: 2418-2429Crossref PubMed Scopus (161) Google Scholar). Similarly, the M153R gene product of myxoma virus ubiquitinates CD4 molecules and causes its lysosomal degradation (11Mansouri M. Bartee E. Gouveia K. Hovey Nerenberg B.T. Barrett J. Thomas L. Thomas G. McFadden G. Fruh K. J. Virol. 2003; 77: 1427-1440Crossref PubMed Scopus (113) Google Scholar). Therefore, viral exploitation of the endosomal sorting machinery for immune evasion appears to be a prevalent strategy. In this report we provide yet another example of a viral protein exploiting the endosomal sorting pathway that may help create an immunosuppressed milieu surrounding the infected cell. The ORF3 protein of hepatitis E virus was observed to associate with TSG101 through the PSAP motif present at its C terminus and expedite the secretion of an immunosuppressant molecule called α1 microglobulin (α1m). α1m is a member of the lipocalin superfamily, found both in blood and in T. T. J. PubMed Scopus Google Scholar). amino from to L. T. A. 2000; PubMed Scopus Google Scholar). α1m is a immunosuppressant and the of of and the and of L. J. PubMed Scopus Google Scholar, E. A. Proc. Natl. Acad. Sci. U. S. A. Scopus Google Scholar, L. PubMed Scopus Google Scholar). in our laboratory have shown that ORF3 with α1m and expedites its and secretion from hepatocytes. This enhanced transport of α1m in ORF3-expressing was to both and The with that the transport is from the of S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In an to the mechanism by which the ORF3 protein be the secretion of α1m, we found the to with PSAP mutant of the ORF3 protein was unable to associate with expedite the secretion of a dominant negative mutant of the VPS4 protein also block the enhanced α1m secretion in type ORF3-expressing the Further, ORF3 was found to simultaneously associate with both α1m and TSG101 in a ternary complex from hepatocyte of suggest that the ORF3 protein associates with TSG101 in a manner analogous to late proteins and expedites the secretion of The of this phenomenon during viral is ORF3 gene of was from S. M. J. Virol. PubMed Google Scholar) and and into the with the ORF3 and and ORF3 have been earlier S. S. J. Virol. PubMed Scopus Google Scholar, S. H. M. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the were as by J. T. Scholar). ORF3 was by two of the mutant were into at the and are and This was as ORF3 from amino to was ORF3 as The product was and into the of ORF3 to get The and are and 101 101 and dominant negative and were from Sundquist O. Garrus J.E. M. Wang Sundquist W.I. J. Cell Biol. 2003; 162: PubMed Scopus Google Scholar, Garrus J.E. S. Sundquist W.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). system III was The study was as J. PubMed Google Scholar). were with activation domain and binding domain and were by them on for for were at and for their ability to on and with to were for by both and as S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Cell and were in with and The were at with to the The were in a or a on and were with and for in and were with of for was α1m was to be The were with and in of with The were and by for at For an of protein was with of at by with of protein for The were three in the in and on by or proteins were by and to The were with for and with and with The proteins were by the to the was as by S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The ORF3 protein was with and α1m or TSG101 was by The was by The were a of was ORF3 of E with in our laboratory have proved that the ORF3 protein of binds to α1m and expedites its transport from the of cultured cells. The observed phenomenon was to both and that the ORF3 protein its at the S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that ORF3 be with crucial of the cellular cargo transport machinery and the pathway the benefit of the virus, as has been observed for many viral gene we the ORF3 protein for any known motifs that with a cellular protein trafficking we observed the of a PSAP motif and amino of the ORF3 that the PSAP motif is different of It was also in which from all The PSAP motif has earlier been to be present in the late domain proteins of many RNA viruses such as HIV-1 and Ebola (2Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). PSAP motif present in late domain proteins has been shown to with TSG101 and the host ESCRT machinery for budding of viral we the ORF3 protein also with was to the ORF3 and of this that a or ORF3 with or TSG101, In addition, the mutant TSG101 as that has been to its ability to bind the PSAP motif Garrus J.E. S. Sundquist W.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was unable to with ORF3 the human the from in the amino of ORF3 we also the ORF3 protein with TSG101 in the system which a Further, the ORF3 protein was unable to associate with the mutant TSG101, thus that the observed was a of the former. This as because the PSAP motif was found in the of the ORF3 protein as a for the of TSG101, which has been earlier J. T. J. Virol. 2003; 77: PubMed Scopus Google was observed in a of and ORF3 with mutant TSG101 as negative in the of the of ORF3 and TSG101 in with as by is also shown in in the ORF3 was found to with type TSG101, which was from in with both ORF3 and Further, of the mutant TSG101 with ORF3 protein was that of type TSG101 with ORF3 and with that of the results from the we a to the association of the ORF3 protein with TSG101 in a cell environment. were with or ORF3 the were with of for and in of were or of the was by and with and the was on by to ORF3 TSG101 was found to be with ORF3 TSG101 ORF3 protein a negative by cell was with and with that ORF3 co-precipitated TSG101 of was to two of and the was and with both ORF3 and TSG101 was thus that both of them as a complex in the cell This that the ORF3 protein associates with TSG101 in a cellular The PSAP of ORF3 for with an to the of the PSAP motif for with the ORF3 we adopted a amino PSAP were to as to the PSAP motif. was to during The gene was into the as a with the activation of the mutant protein was an in system of the PSAP mutant ORF3 gene in the was by the the activation domain and the ORF3 thus a type ORF3 in was used as a in the system was by the protein machinery present in the The protein thus was with type or PSAP mutant ORF3 protein were and the of ORF3 protein was by Both type and protein were and and by the The protein at a the type protein because of the of the to the former. not any to the ORF3 protein thus that the observed were to the ORF3 The protein was for with TSG101 in a this protein lost its ability to with TSG101 a we also the ability of two of ORF3 gene to with These were used earlier in S. S. J. Virol. PubMed Scopus Google Scholar). In with our proposed the ORF3 amino of the ORF3 protein not with the TSG101 the amino was of with TSG101 as as the type ORF3 protein of the any with the TSG101 mutant of the ORF3 protein was a for its association with It has been earlier that the ORF3 protein is at a at the amino from the of the this to of ORF3 M. S. J. Virol. PubMed Google Scholar). used this ORF3 mutant S. H. M. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) as a to its with The ORF3 protein with TSG101 as by the by as as Therefore, the of the ORF3 protein with TSG101 is of the of The were by a The PSAP mutant ORF3 gene was into the is of the protein in was by of the cell not were with type ORF3 or PSAP mutant ORF3 with and by the protein was unable to associate with TSG101, type the TSG101 product and The the of the ORF3 protein in This was by the the of ORF3 protein that be in TSG101 the type ORF3 protein to of protein a as a to the of that the PSAP motif of the ORF3 protein is and the ORF3 protein the ability to associate with ORF3 with the TSG101 proved that type ORF3 and the TSG101 protein associate with in a cellular we subsequently the two proteins with were cultured on and with the type ORF3 or the were and for of the ORF3 protein was by with and and that of TSG101 was by with II, and the type ORF3 protein with TSG101, the mutant was unable to do with in the of a of FITC, as in the of type ORF3 protein in the of the was observed In type ORF3-expressing TSG101 was found in the in the was and a was present in the in mutant was observed from that of cells. The PSAP of ORF3 for of α1 from ORF3-expressing that association with TSG101 is a of the ORF3 TSG101 is a in of the ESCRT we that by with ORF3 be the of the ESCRT which may α1m as a to its This was on that association of a viral late domain protein HIV-1 with TSG101 is able to ESCRT machinery to virus budding. we this enables the ORF3 protein to expedite the secretion of this we the protein in and α1m from as with type ORF3-expressing cells. is shown in ORF3 and α1m are with and FITC, type ORF3 the α1m as earlier S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and ORF3-expressing are with and that as are with In the α1m was The protein of α1m in the was with that of and This that the ability of the ORF3 protein to expedite the secretion of α1m from was a by the PSAP motif present within the ORF3 of two were for and α1m was This provided a of the observed phenomenon of the type ORF3 protein the In the mutant ORF3 the phenomenon was of the protein The this this may be to the of α1m regulated by with ORF3 or PSAP mutant ORF3 were at and for in to of were in different ORF3 protein were for α1m of α1 microglobulin were as The shown are of three were from the of ORF3 the of type mutant of of of in a the we the protein of α1m from type ORF3 or were with ORF3 or the were with and both the and the were was with and the cell was shown in the of the type ORF3 in α1m protein in the this phenomenon was not observed in the The a of of α1m as from three of the The that the ORF3 protein the ESCRT complex by binding to TSG101 as to enhance the trafficking of α1m from the this we α1m secretion in type ORF3-expressing a dominant negative mutant of VPS4 is an family that at the of the endosomal sorting pathway by the of different from the This enables of the cargo from the An mutant of VPS4 is and thus as a dominant negative mutant the type VPS4 protein M. EMBO J. PubMed Scopus Google Scholar, M. EMBO J. PubMed Scopus Google Scholar). for the VPS4 dominant negative mutant with type ORF3 was that ORF3 exploits the endosomal sorting pathway to enhance the secretion of α1m, the VPS4 dominant negative mutant be able to block because VPS4 of TSG101 in this this were with type ORF3 and type or dominant negative mutant of VPS4 a and and are the two of VPS4 in A. S. S. A. K. 2003; PubMed Scopus Google The were with for and the of α1m were by shown in and the the of α1m were found to be in the dominant negative and the dominant negative not a in the of α1m protein and of ORF3 in was by the cell with of type and dominant negative and was by a because enhanced protein and a to α1m secretion in normal is regulated by VPS4 we simultaneously with type or dominant negative mutant of and and the of was in the of α1m in dominant negative mutant was observed in the dominant negative a cell was The was α1m to that of this appears that be in α1m secretion in the normal of its the of ORF3 this process. of TSG101 in the endosomal sorting we that ORF3 the endosomal sorting machinery for the secretion of It be that ORF3 as an that the α1m and the ESCRT in enhanced transport and secretion of this we to the complex from human cells. ORF3-expressing were with with and of the was The was on by of ORF3 protein The α1m was and with to the of TSG101 shown in the of the both type ORF3 and associate with was unable to TSG101 The that all TSG101, and α1m, were a ternary complex was by the of both α1m and TSG101 proteins from with Therefore, we that the ORF3 protein simultaneously binds to both α1m and TSG101 and the of the ESCRT complex. This to enhanced trafficking and secretion of α1m in ORF3-expressing cells. In the present study, we report that the ORF3 protein of the hepatitis E virus associates with TSG101 and exploits the cellular ESCRT machinery to expedite the secretion of an immunosuppressant α1m, from the human cells. This study a to our earlier that the ORF3 protein with α1m and expedites its transport from the of cells. In addition, this study also a novel example of a viral protein the endosomal sorting pathway to enhance the secretion of an immunosuppressant which may help in viral during its of The that the observed phenomenon was not an of the was in many For example, a PSAP mutant of the ORF3 protein was unable to associate with TSG101 and of the secretion of α1 microglobulin; a dominant negative mutant of VPS4 which of TSG101 in the endosomal sorting block the of type the observed phenomenon is a of the ORF3 The ORF3 protein is from the of the hepatitis E is in hepatitis E the role of ORF3 protein during viral is is a It causes hepatitis in of the any to its have been for In to the of a system or in infection system has to its viral life infection in has been to be with a because of is important to study the life cycle and of this ORF3 has earlier been shown to be a that associates with the M. S. J. Virol. PubMed Google Scholar). It also with the major capsid protein of the virus S. H. M. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google on has been to be a of the virus for this Further, has been shown to with different cell to of the ORF3 protein H. S. D. S. M. M. L. D. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In an to the of this a human was ORF3 protein as in a system in our This study ORF3 to with (α1 microglobulin as as its product was observed that of the ORF3 protein the secretion of α1m from the on which was proposed that the ORF3 protein may help maintain an immunosuppressed milieu surrounding the infected S. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a mechanism for this process in this we that this phenomenon be during the of an virus system to be to this viral gene have earlier been shown to bind TSG101, which is a central of the ESCRT TSG101 recognition motif the PSAP is present in late domains of many RNA virus capsid proteins such as that of and virus (2Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). It has been that the PSAP motif of proteins the ESCRT machinery by binding TSG101, which is essential for viral budding. of ESCRT machinery for the transport of cellular proteins by viral has not been the ORF3 protein the example of a viral protein that the ESCRT machinery for cellular protein viral proteins have earlier been to exploit the endosomal sorting pathway to host immune One example of major histocompatibility complex class I by the k3 gene product of Kaposi virus (10Hewitt E.W. Duncan L. Mufti D. Baker J. Stevenson P.G. Lehner P.J. EMBO J. 2002; 21: 2418-2429Crossref PubMed Scopus (161) Google Scholar). a different of appears to a by an immunosuppressed TSG101 is known to both ubiquitinated and of Gag protein has been shown to enhance its binding with the VP40 protein of Ebola virus was shown to enhance viral budding both by association with TSG101 through a PSAP motif or of TSG101 by the PPXY motif in a manner (3Licata J.M. Simpson-Holley M. Wright N.T. Han Z. Paragas J. Harty R.N. J. Virol. 2003; 77: 1812-1819Crossref PubMed Scopus (231) Google Scholar). of late domain proteins is by the of a PPXY motif in them that with the Nedd4 family of ubiquitin ligase and found in the VP40 gene product of Ebola virus, the ORF3 protein also an the tyrosine is with that this motif may bind to WW domain present in Nedd4 family of ubiquitin we to a of ORF3 protein in human cell be attributed to of our system or of the ORF3 It may also be that the of is and regulated in the of the ORF3 to in may help the motif is in ORF3 of the VPS4 protein in known as and the dominant negative mutant of block the of ORF3 on we do not have a for this observation, be that ORF3 of α1m One that our from this study is α1m secretion is the for which ORF3 protein a PSAP motif. It is important to that this motif is all of human and HEV, the motif is In all RNA the PSAP motif is present in the capsid in budding of the may be that in to α1m ORF3 as an and its association with TSG101 the ESCRT machinery for budding of a system this Sundquist of for the of TSG101 and its mutant in and for the VPS4 type and dominant negative mutant for the for help in

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.