Abstract
The membrane glycoproteins G1 and G2 of the members of the Bunyaviridae family are synthesized as a precursor from a single open reading frame. Here, we have analyzed the processing and membrane insertion of G1 and G2 of a member of the Phlebovirus genus, Uukuniemi virus. By expressing C-terminally truncated forms of the p10 precursor containing the whole of G1 and decreasing portions of G2, we found that processing in BHK21 cells occurred with an efficiency of about 50% if G1 was followed by 50 residues of G2, while complete processing occurred if 98, 150, or 200 residues of G2 were present. Surprisingly, processing of all truncated G2 forms was less efficient in HeLa cells. Proteinase K treatment of microsomes isolated from infected cells indicated that the C terminus of G1 is exposed on the cytoplasmic face. Using G1 tail peptide antisera, the tail was likewise found by immunofluorescence to be exposed on the cytoplasmic face in streptolysin O-permeabilized cells. By introducing stop codons at various positions of the G1 tail and at the natural cleavage site between G1 and G2 and expressing these mutants in BHK cells, we found that no further processing of the G1 C terminus occurred following cleavage of G2 by the signal peptidase. This was also supported by the finding that an antiserum raised against a peptide corresponding to the region immediately upstream from the G2 signal sequence reacted in immunoblotting with G1 from virions. Finally, we show that both G1 and G2 are palmitylated. Taken together, these results show that processing of p10 of Uukuniemi virus occurs cotranslationally at only one site, i.e., downstream of the internal G2 signal sequence. G1 and G2 are inserted as type I proteins into the lipid bilayer, leaving the G1 tail exposed on the cytoplasmic face of the membrane. Since the G2 tail is only 5 residues long, the G1 tail is likely to be responsible for the interaction with the nucleoproteins during the budding process, in addition to harboring a Golgi localization signal.
Full Text
The Full Text of this article is available as a PDF (390.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams G. A., Rose J. K. Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell. 1985 Jul;41(3):1007–1015. doi: 10.1016/s0092-8674(85)80081-7. [DOI] [PubMed] [Google Scholar]
- Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
- Elliott R. M. Molecular biology of the Bunyaviridae. J Gen Virol. 1990 Mar;71(Pt 3):501–522. doi: 10.1099/0022-1317-71-3-501. [DOI] [PubMed] [Google Scholar]
- Elliott R. M., Schmaljohn C. S., Collett M. S. Bunyaviridae genome structure and gene expression. Curr Top Microbiol Immunol. 1991;169:91–141. doi: 10.1007/978-3-642-76018-1_4. [DOI] [PubMed] [Google Scholar]
- Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fazakerley J. K., Gonzalez-Scarano F., Strickler J., Dietzschold B., Karush F., Nathanson N. Organization of the middle RNA segment of snowshoe hare Bunyavirus. Virology. 1988 Dec;167(2):422–432. [PubMed] [Google Scholar]
- Fiedler K., Simons K. Characterization of VIP36, an animal lectin homologous to leguminous lectins. J Cell Sci. 1996 Jan;109(Pt 1):271–276. doi: 10.1242/jcs.109.1.271. [DOI] [PubMed] [Google Scholar]
- Kamrud K. I., Schmaljohn C. S. Expression strategy of the M genome segment of Hantaan virus. Virus Res. 1994 Jan;31(1):109–121. doi: 10.1016/0168-1702(94)90074-4. [DOI] [PubMed] [Google Scholar]
- Kormelink R., de Haan P., Meurs C., Peters D., Goldbach R. The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments. J Gen Virol. 1992 Nov;73(Pt 11):2795–2804. doi: 10.1099/0022-1317-73-11-2795. [DOI] [PubMed] [Google Scholar]
- Kuismanen E., Bång B., Hurme M., Pettersson R. F. Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. J Virol. 1984 Jul;51(1):137–146. doi: 10.1128/jvi.51.1.137-146.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuismanen E., Hedman K., Saraste J., Pettersson R. F. Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Mol Cell Biol. 1982 Nov;2(11):1444–1458. doi: 10.1128/mcb.2.11.1444. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuismanen E. Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2. J Virol. 1984 Sep;51(3):806–812. doi: 10.1128/jvi.51.3.806-812.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuismanen E., Saraste J., Pettersson R. F. Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex. J Virol. 1985 Sep;55(3):813–822. doi: 10.1128/jvi.55.3.813-822.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lahtinen U., Hellman U., Wernstedt C., Saraste J., Pettersson R. F. Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein. J Biol Chem. 1996 Feb 23;271(8):4031–4037. doi: 10.1074/jbc.271.8.4031. [DOI] [PubMed] [Google Scholar]
- Lappin D. F., Nakitare G. W., Palfreyman J. W., Elliott R. M. Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm. J Gen Virol. 1994 Dec;75(Pt 12):3441–3451. doi: 10.1099/0022-1317-75-12-3441. [DOI] [PubMed] [Google Scholar]
- Liljeström P., Garoff H. Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J Virol. 1991 Jan;65(1):147–154. doi: 10.1128/jvi.65.1.147-154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu N., Brown D. T. Phosphorylation and dephosphorylation events play critical roles in Sindbis virus maturation. Virology. 1993 Oct;196(2):703–711. doi: 10.1006/viro.1993.1527. [DOI] [PubMed] [Google Scholar]
- Liu N., Brown D. T. Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes. J Cell Biol. 1993 Feb;120(4):877–883. doi: 10.1083/jcb.120.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marriott A. C., el-Ghorr A. A., Nuttall P. A. Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy. Virology. 1992 Oct;190(2):606–615. doi: 10.1016/0042-6822(92)90898-y. [DOI] [PubMed] [Google Scholar]
- Melin L., Persson R., Andersson A., Bergström A., Rönnholm R., Pettersson R. F. The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex. Virus Res. 1995 Apr;36(1):49–66. doi: 10.1016/0168-1702(95)00006-C. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Naeve C. W., Williams D. Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion. EMBO J. 1990 Dec;9(12):3857–3866. doi: 10.1002/j.1460-2075.1990.tb07604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Persson R., Pettersson R. F. Formation and intracellular transport of a heterodimeric viral spike protein complex. J Cell Biol. 1991 Jan;112(2):257–266. doi: 10.1083/jcb.112.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pettersson R. F., Andersson A., Melin L. Mapping a retention signal for Golgi localization of a viral spike protein complex. Cold Spring Harb Symp Quant Biol. 1995;60:147–155. doi: 10.1101/sqb.1995.060.01.018. [DOI] [PubMed] [Google Scholar]
- Pettersson R. F. Protein localization and virus assembly at intracellular membranes. Curr Top Microbiol Immunol. 1991;170:67–106. doi: 10.1007/978-3-642-76389-2_3. [DOI] [PubMed] [Google Scholar]
- Pettersson R., Käriäinen L. The ribonucleic acids of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology. 1973 Dec;56(2):608–619. doi: 10.1016/0042-6822(73)90062-7. [DOI] [PubMed] [Google Scholar]
- Rönkä H., Hildén P., Von Bonsdorff C. H., Kuismanen E. Homodimeric association of the spike glycoproteins G1 and G2 of Uukuniemi virus. Virology. 1995 Aug 1;211(1):241–250. doi: 10.1006/viro.1995.1397. [DOI] [PubMed] [Google Scholar]
- Rönnholm R., Pettersson R. F. Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2. Virology. 1987 Sep;160(1):191–202. doi: 10.1016/0042-6822(87)90060-2. [DOI] [PubMed] [Google Scholar]
- Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Suomalainen M., Garoff H., Baron M. D. The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro. J Virol. 1990 Nov;64(11):5500–5509. doi: 10.1128/jvi.64.11.5500-5509.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ulmanen I., Seppälä P., Pettersson R. F. In vitro translation of Uukuniemi virus-specific RNAs: identification of a nonstructural protein and a precursor to the membrane glycoproteins. J Virol. 1981 Jan;37(1):72–79. doi: 10.1128/jvi.37.1.72-79.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yonath A., Leonard K. R., Wittmann H. G. A tunnel in the large ribosomal subunit revealed by three-dimensional image reconstruction. Science. 1987 May 15;236(4803):813–816. doi: 10.1126/science.3576200. [DOI] [PubMed] [Google Scholar]