Abstract
To determine whether there is a cis-acting effect of translational expression of gag on RNA encapsidation, we compared the encapsidation of wild-type RNA with that of a mutant in which the translation of gag was ablated. This comparison indicated that there is not such a cis effect. To determine what is necessary and sufficient for encapsidation, we measured the relative encapsidation efficiencies of human immunodeficiency virus type 1 vector RNAs containing mutations in domains proximal to the canonical encapsidation signal or containing large deletions in the remainder of the genome. These data indicate that TAR and two additional regions are required for encapsidation and that the 5' end of the genome is sufficient for encapsidation. The Rev-responsive element is required mainly for efficient RNA transport from the nucleus to the cytoplasm. A foreign sequence was found to have a negative effect on encapsidation upon placement within the parental vector. Interestingly, this negative effect was compounded by multiple copies of the sequence.
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- Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aiyar A., Cobrinik D., Ge Z., Kung H. J., Leis J. Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription. J Virol. 1992 Apr;66(4):2464–2472. doi: 10.1128/jvi.66.4.2464-2472.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Awang G., Sen D. Mode of dimerization of HIV-1 genomic RNA. Biochemistry. 1993 Oct 26;32(42):11453–11457. doi: 10.1021/bi00093a024. [DOI] [PubMed] [Google Scholar]
- Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
- Berkhout B., Klaver B., Das A. T. A conserved hairpin structure predicted for the poly(A) signal of human and simian immunodeficiency viruses. Virology. 1995 Feb 20;207(1):276–281. doi: 10.1006/viro.1995.1077. [DOI] [PubMed] [Google Scholar]
- Berkhout B., van Wamel J., Klaver B. Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions. J Mol Biol. 1995 Sep 8;252(1):59–69. doi: 10.1006/jmbi.1994.0475. [DOI] [PubMed] [Google Scholar]
- Berkowitz R. D., Goff S. P. Analysis of binding elements in the human immunodeficiency virus type 1 genomic RNA and nucleocapsid protein. Virology. 1994 Jul;202(1):233–246. doi: 10.1006/viro.1994.1339. [DOI] [PubMed] [Google Scholar]
- Berkowitz R. D., Hammarskjöld M. L., Helga-Maria C., Rekosh D., Goff S. P. 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology. 1995 Oct 1;212(2):718–723. doi: 10.1006/viro.1995.1530. [DOI] [PubMed] [Google Scholar]
- Berkowitz R. D., Luban J., Goff S. P. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol. 1993 Dec;67(12):7190–7200. doi: 10.1128/jvi.67.12.7190-7200.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berthold E., Maldarelli F. cis-acting elements in human immunodeficiency virus type 1 RNAs direct viral transcripts to distinct intranuclear locations. J Virol. 1996 Jul;70(7):4667–4682. doi: 10.1128/jvi.70.7.4667-4682.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bray M., Prasad S., Dubay J. W., Hunter E., Jeang K. T., Rekosh D., Hammarskjöld M. L. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1256–1260. doi: 10.1073/pnas.91.4.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buchschacher G. L., Jr, Panganiban A. T. Human immunodeficiency virus vectors for inducible expression of foreign genes. J Virol. 1992 May;66(5):2731–2739. doi: 10.1128/jvi.66.5.2731-2739.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Böhnlein S., Hauber J., Cullen B. R. Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat. J Virol. 1989 Jan;63(1):421–424. doi: 10.1128/jvi.63.1.421-424.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clever J. L., Wong M. L., Parslow T. G. Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA. J Virol. 1996 Sep;70(9):5902–5908. doi: 10.1128/jvi.70.9.5902-5908.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clever J., Sassetti C., Parslow T. G. RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. J Virol. 1995 Apr;69(4):2101–2109. doi: 10.1128/jvi.69.4.2101-2109.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
- Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
- Embretson J. E., Temin H. M. Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line. J Virol. 1987 Sep;61(9):2675–2683. doi: 10.1128/jvi.61.9.2675-2683.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feng S., Holland E. C. HIV-1 tat trans-activation requires the loop sequence within tar. Nature. 1988 Jul 14;334(6178):165–167. doi: 10.1038/334165a0. [DOI] [PubMed] [Google Scholar]
- Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
- Fridell R. A., Fischer U., Lührmann R., Meyer B. E., Meinkoth J. L., Malim M. H., Cullen B. R. Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2936–2940. doi: 10.1073/pnas.93.7.2936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geigenmüller U., Linial M. L. Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence. J Virol. 1996 Jan;70(1):667–671. doi: 10.1128/jvi.70.1.667-671.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goodarzi G., Im G. J., Brackmann K., Grandgenett D. Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase. J Virol. 1995 Oct;69(10):6090–6097. doi: 10.1128/jvi.69.10.6090-6097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrich D., Mavankal G., Mette-Snider A., Gaynor R. B. Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication. J Virol. 1995 Aug;69(8):4906–4913. doi: 10.1128/jvi.69.8.4906-4913.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrich D., Ulich C., Gaynor R. B. A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription. J Virol. 1996 Jun;70(6):4017–4027. doi: 10.1128/jvi.70.6.4017-4027.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holland S. M., Ahmad N., Maitra R. K., Wingfield P., Venkatesan S. Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure. J Virol. 1990 Dec;64(12):5966–5975. doi: 10.1128/jvi.64.12.5966-5975.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
- Kao S. Y., Calman A. F., Luciw P. A., Peterlin B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987 Dec 3;330(6147):489–493. doi: 10.1038/330489a0. [DOI] [PubMed] [Google Scholar]
- Kaye J. F., Lever A. M. trans-acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1. J Virol. 1996 Feb;70(2):880–886. doi: 10.1128/jvi.70.2.880-886.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaye J. F., Richardson J. H., Lever A. M. cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging. J Virol. 1995 Oct;69(10):6588–6592. doi: 10.1128/jvi.69.10.6588-6592.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim H. J., Lee K., O'Rear J. J. A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA. Virology. 1994 Jan;198(1):336–340. doi: 10.1006/viro.1994.1037. [DOI] [PubMed] [Google Scholar]
- Laughrea M., Jetté L. A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA. Biochemistry. 1994 Nov 15;33(45):13464–13474. doi: 10.1021/bi00249a035. [DOI] [PubMed] [Google Scholar]
- Levin J. G., Seidman J. G. Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA. J Virol. 1979 Jan;29(1):328–335. doi: 10.1128/jvi.29.1.328-335.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Litvak S., Sarih-Cottin L., Fournier M., Andreola M., Tarrago-Litvak L. Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. Trends Biochem Sci. 1994 Mar;19(3):114–118. doi: 10.1016/0968-0004(94)90203-8. [DOI] [PubMed] [Google Scholar]
- Luban J., Goff S. P. Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J Virol. 1991 Jun;65(6):3203–3212. doi: 10.1128/jvi.65.6.3203-3212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Luban J., Goff S. P. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994 Jun;68(6):3784–3793. doi: 10.1128/jvi.68.6.3784-3793.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mak J., Jiang M., Wainberg M. A., Hammarskjöld M. L., Rekosh D., Kleiman L. Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol. 1994 Apr;68(4):2065–2072. doi: 10.1128/jvi.68.4.2065-2072.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
- Marquet R., Paillart J. C., Skripkin E., Ehresmann C., Ehresmann B. Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site. Nucleic Acids Res. 1994 Jan 25;22(2):145–151. doi: 10.1093/nar/22.2.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McBride M. S., Panganiban A. T. Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo. J Virol. 1997 Mar;71(3):2050–2058. doi: 10.1128/jvi.71.3.2050-2058.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McBride M. S., Panganiban A. T. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol. 1996 May;70(5):2963–2973. doi: 10.1128/jvi.70.5.2963-2973.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
- Muriaux D., Girard P. M., Bonnet-Mathonière B., Paoletti J. Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide. J Biol Chem. 1995 Apr 7;270(14):8209–8216. doi: 10.1074/jbc.270.14.8209. [DOI] [PubMed] [Google Scholar]
- Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagashunmugam T., Velpandi A., Goldsmith C. S., Zaki S. R., Kalyanaraman V. S., Srinivasan A. Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4114–4118. doi: 10.1073/pnas.89.9.4114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okamoto T., Wong-Staal F. Demonstration of virus-specific transcriptional activator(s) in cells infected with HTLV-III by an in vitro cell-free system. Cell. 1986 Oct 10;47(1):29–35. doi: 10.1016/0092-8674(86)90363-6. [DOI] [PubMed] [Google Scholar]
- Paillart J. C., Marquet R., Skripkin E., Ehresmann B., Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA. J Biol Chem. 1994 Nov 4;269(44):27486–27493. [PubMed] [Google Scholar]
- Parolin C., Dorfman T., Palú G., Göttlinger H., Sodroski J. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes. J Virol. 1994 Jun;68(6):3888–3895. doi: 10.1128/jvi.68.6.3888-3895.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peters G. G., Hu J. Reverse transcriptase as the major determinant for selective packaging of tRNA's into Avian sarcoma virus particles. J Virol. 1980 Dec;36(3):692–700. doi: 10.1128/jvi.36.3.692-700.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richardson J. H., Child L. A., Lever A. M. Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region. J Virol. 1993 Jul;67(7):3997–4005. doi: 10.1128/jvi.67.7.3997-4005.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rizvi T. A., Panganiban A. T. Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. J Virol. 1993 May;67(5):2681–2688. doi: 10.1128/jvi.67.5.2681-2688.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sakaguchi K., Zambrano N., Baldwin E. T., Shapiro B. A., Erickson J. W., Omichinski J. G., Clore G. M., Gronenborn A. M., Appella E. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5219–5223. doi: 10.1073/pnas.90.11.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sherman P. A., Dickson M. L., Fyfe J. A. Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions. J Virol. 1992 Jun;66(6):3593–3601. doi: 10.1128/jvi.66.6.3593-3601.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Skripkin E., Paillart J. C., Marquet R., Ehresmann B., Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4945–4949. doi: 10.1073/pnas.91.11.4945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sundquist W. I., Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3393–3397. doi: 10.1073/pnas.90.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabernero C., Zolotukhin A. S., Valentin A., Pavlakis G. N., Felber B. K. The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J Virol. 1996 Sep;70(9):5998–6011. doi: 10.1128/jvi.70.9.5998-6011.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vicenzi E., Dimitrov D. S., Engelman A., Migone T. S., Purcell D. F., Leonard J., Englund G., Martin M. A. An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. J Virol. 1994 Dec;68(12):7879–7890. doi: 10.1128/jvi.68.12.7879-7890.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wakefield J. K., Kang S. M., Morrow C. D. Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His). J Virol. 1996 Feb;70(2):966–975. doi: 10.1128/jvi.70.2.966-975.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wakefield J. K., Morrow C. D. Mutations within the primer binding site of the human immunodeficiency virus type 1 define sequence requirements essential for reverse transcription. Virology. 1996 Jun 15;220(2):290–298. doi: 10.1006/viro.1996.0317. [DOI] [PubMed] [Google Scholar]
- Wakefield J. K., Wolf A. G., Morrow C. D. Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys). J Virol. 1995 Oct;69(10):6021–6029. doi: 10.1128/jvi.69.10.6021-6029.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]