Sequence variation of the human ... - Semantic Scholar
Journal of General Virology (1997), 78, 837–840. Printed in Great Britain ...............................................................................................................................................................................................................
SHORT COMMUNICATION
Sequence variation of the human immunodeficiency virus primer-binding site suggests the use of an alternative tRNALys molecule in reverse transcription Atze T. Das, Bep Klaver and Ben Berkhout Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3« end of this tRNA and a sequence near the 5« end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNALys3 as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNALys is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNALys5 and differs from tRNALys3 at five nucleotide positions. These results suggest that HIV uses both tRNALys3 and tRNALys5 molecules as primer, causing a switch of the PBS sequence.
The replication cycle of retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA, which is integrated into the host-cell genome (reviewed by Varmus & Swanstrom, 1984). This process is mediated by the virion-associated enzyme reverse transcriptase (RT) and a cellular tRNA is used as primer. This tRNA binds with its 3«terminal 18 nucleotides to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). Apart from the complementarity between the PBS and tRNA sequences, additional basepairing interactions between the tRNA primer and vRNA template may support this binding (Isel et al., 1995 ; Berkhout & Schoneveld, 1993 ; Aiyar et al., 1994). Furthermore, binding of the tRNA primer onto the Author for correspondence : Ben Berkhout Fax 31 20 6916531. e-mail B.Berkhout!AMC.UVA.NL
0001-4426 # 1997 SGM
vRNA can be activated by RT and nucleocapsid (NC) protein (Araya et al., 1979 ; Prats et al., 1988 ; Barat et al., 1989 ; Li et al., 1996). The RT protein, or the precursor Gag-Pol protein, was also suggested to be involved in selective encapsidation of the tRNA primer into virions (Peters & Hu, 1980 ; Levin & Seidman, 1981 ; Mak et al., 1994). The human immunodeficiency viruses (HIV-1 and HIV-2) and all simian immunodeficiency viruses efficiently replicate with tRNALys$ as primer. By mutating the PBS sequence, we (Das et al., 1995) and others (Li et al., 1994 ; Wakefield et al., 1995) recently demonstrated that HIV-1 can replicate with other tRNA primers (tRNAIle, tRNAHis, tRNALys",#, tRNAPhe, tRNAPro, tRNATrp), although less efficiently compared with the natural tRNALys$ primer. These mutants are unstable and revert to the wild-type PBSLys$ sequence upon prolonged culturing (Das et al., 1995). This reversion is mediated by annealing of a wild-type tRNALys$ onto the mutant PBS sequence, followed by copying of the 18 nucleotides at the 3« terminus of this tRNA primer during reverse transcription. During this mutation}reversion analysis, we occasionally observed a variant PBS sequence with a single C ! U substitution in the centre of the motif (Fig. 1). This suggests the presence of a variant tRNA primer resembling tRNALys$ in human cells. The PBS point mutation corresponds with a G ! A substitution at position 69 in the tRNA acceptor-stem (Fig. 2). A mouse gene encoding such a putative tRNALys species was previously described (Han & Harding, 1983). This murine tRNA contains five nucleotide differences compared with the human tRNALys$ sequence (positions 4, 15, 17, 48 and 69). We arbitrarily refer to this tRNA as tRNALys& (Fig. 2). The PBSLys& sequence was also observed in other mutation}reversion studies (Vicenzi et al., 1994 ; Das & Berkhout, 1995 ; A. T. Das and others, unpublished). Alignment of the PBS region of natural HIV-1 and HIV-2 isolates demonstrates considerable sequence variation up- and downstream of the PBS sequence, but relatively little variation is observed in the 18 nt PBS sequence (Fig. 3 ; Myers et al., 1994). Three out of the 24 sequenced HIV-2 isolates, but none of the 30 sequenced HIV-1 isolates, have a PBSLys& sequence. We tested the stability of the PBSLys& sequence in the HIV-2
IDH
A. T. Das, B. Klaver and B. Berkhout
Fig. 1. Primer-binding sites in HIV. PBS sequences (lower lines) complementary to tRNALys3 and tRNALys5 (upper lines) are found in HIV-1 and HIV-2 viruses, both natural isolates (Fig. 3) and viruses obtained in mutation/reversion studies. Nucleotides in the PBSLys5/tRNALys5 duplex that differ from the PBSLys3/tRNALys3 duplex are marked by a shaded box. Other PBS sequences that are not perfectly complementary to either tRNALys3 or tRNALys5 are not shown (n ¯ 2 for HIV-1 natural isolates, n ¯ 4 for HIV-2 natural isolates, n ¯ 3 for HIV-1 laboratory isolates). These variants may have resulted from PCR or sequencing errors. a, Combined results of studies in which the PBS site (Das & Berkhout, 1995 ; Das et al., 1995), the R-U5 region (A. T. Das and others, unpublished) and the U5 region (Vicenzi et al., 1994) were mutated.
Fig. 2. Secondary structure of tRNALys molecules. The cloverleaf structures of tRNALys5 (Han & Harding, 1983), tRNALys3, tRNALys1 and tRNALys2 are shown. tRNALys4 (not shown) is a tRNALys form specific for transformed cells that was initially thought to be an undermodified tRNALys2 (Raba et al., 1979), but turned out to be a differentially modified form of tRNALys2 (Hayenga et al., 1986). Nucleotides differing from tRNALys3 are shaded. Base modifications are indicated according to standard nomenclature (Sprinzl et al., 1989) ; A1, 1-methyladenosine ; A7, N-((9-β-D-ribofuranosylpurine-6-yl)carbamoyl)threonine ; A9, N-((9-β-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine ; C5, 5-methylcytidine ; D, dihydrouridine ; G2, N2-methylguanosine ; G7, 7-methylguanosine ; T3, 2«-O-methyl-5-methyluridine ; U9, 5-methoxycarbonylmethyl-2-thiouridine ; Ψ, pseudouridine.
isolate ROD (Peden & Martin, 1996). SupT1 T cells were transfected with the pROD10 proviral clone and cultured for 2 weeks as previously described (Das et al., 1995). The viruscontaining culture supernatant was used to infect fresh cells, which were cultured for another week. To determine the PBS sequence of the viral progeny, the LTR-leader region of the proviral DNA was amplified from total cellular DNA by PCR and cloned as described (Das et al., 1995). Four clones were sequenced and all contained a PBS sequence complementary to tRNALys$ instead of tRNALys&, indicating that the PBSLys& sequence is not stably maintained in the HIV-2 isolate. These combined results suggest that the variant tRNALys& molecule is used at a low frequency (approximately 3 %, Fig. 1) in reverse transcription by the HIV virus.
IDI
There are several possible explanations for the infrequent use of tRNALys& as primer in HIV replication. First and most importantly, this tRNA species may represent a so-called minor tRNA. Indeed, tRNALys& is not one of the three major tRNALys species identified in rabbit liver (tRNALys", tRNALys#, tRNALys$ ; Raba et al., 1979), and it is also not one of the four major tRNA species identified in HIV-1 virions (tRNALys", tRNALys#, tRNALys$, tRNAIle ; Jiang et al., 1993). Alternatively, inefficient packaging of tRNALys& may result in a low concentration of this tRNA species in virions, and thus in an infrequent priming with this tRNA during HIV replication. We note that the tRNALys" and tRNALys# variants, which have 15 and 13 base changes compared with tRNALys$, are packaged into HIV-1 virions (Jiang et al., 1993). Differences
tRNA primers in HIV replication
Fig. 3. PBS sequences of HIV-1 and HIV-2 isolates. The PBS sequences of HIV-1 and HIV-2 isolates are grouped according to the different subtypes (Myers et al., 1994). ^, deleted nucleotide ; y, insertion of ACTTGACGGTAATAGG (in the HIV-1 IBNG isolate). Three of the HIV-2 isolates have a deletion of one G-nucleotide in the PBS sequence. This may indicate that yet another tRNALys variant with a deletion of one C-nucleotide was used as primer for reverse transcription.
in PBS-annealing efficiency may also result in reduced tRNALys& usage. tRNALys$ and tRNALys& differ at five nucleotide positions (Fig. 2). The putative additional tRNAvRNA contacts involving the anticodon loop (Isel et al., 1995) will not be different for these two tRNA species, but one of the nucleotide changes (at position 69) affects the basepairing between the tRNA and the PBS (Fig. 1). At this position, tRNALys$ can form a G-C basepair with PBSLys$ and a G-U basepair with PBSLys&. In contrast, tRNALys& forms an A-U basepair with PBSLys& and an A-C mismatch with PBSLys$. Thus, annealing of tRNALys$ may be more efficient, resulting in the accumulation of PBSLys$ sites. Finally, the more frequent tRNALys$ usage may result from a more efficient priming by the HIV-1 RT enzyme. We note that this initiation reaction is rather specific because the related tRNALys" and tRNALys# primers are not efficiently used by the HIV-1 enzyme (Oude Essink et al., 1996). Consistent with this result, spontaneous PBSLys",# variation is never observed (Figs 1 and 3). Concluding, the occurrence of a PBSLys& sequence in HIV retroviruses suggests that a tRNALys& molecule is present in human cells. This minor tRNALys& species may play a specialized role in the metabolism of the host cell, but there is
currently no evidence for a specific role of this variant tRNA primer in HIV biology. We thank Wim van Est for artwork and Keith Peden for the gift of the HIV-1 pLAI and HIV-2 pROD10 proviral clones. This work was supported by the Dutch Cancer Society (KWF).
References Aiyar, A., Ge, Z. & Leis, J. (1994). A specific orientation of RNA secondary structures is required for initiation of reverse transcription. Journal of Virology 68, 611–618. Araya, A., Sarih, L. & Litvak, S. (1979). Reverse transcriptase mediated binding of primer tRNA to the viral genome. Nucleic Acids Research 6, 3831–3843. Barat, C., Lullien, V., Schatz, O., Keith, G., Mugeyre, M. T., Gru$ ningerLeitch, F., Barre! -Sinoussi, F., LeGrice, S. F. J. & Darlix, J. L. (1989).
HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO Journal 11, 3279–3285. Berkhout, B. & Schoneveld, I. (1993). Secondary structure of the HIV2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Research 21, 1171–1178.
IDJ
A. T. Das, B. Klaver and B. Berkhout Das, A. T. & Berkhout, B. (1995). Efficient extension of a misaligned
tRNA-primer during replication of the HIV-1 retrovirus. Nucleic Acids Research 23, 1319–1326. Das, A. T., Klaver, B. & Berkhout, B. (1995). Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). Journal of Virology 69, 3090–3097. Han, J. H. & Harding, J. D. (1983). Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis – nucleotide sequence of a mouse lysine tRNA gene. Nucleic Acids Research 11, 2053–2064. Hayenga, K., Hedgcoth, C., Harrison, M., Lin, V. K. & Ortwerth, B. J. (1986). Structural relationship between tRNA(Lys2) and tRNA(Lys4)
from mouse lymphoma cells. Molecular and Cellular Biochemistry 71, 25–30. Isel, C., Ehresmann, C., Keith, G., Ehresmann, B. & Marquet, R. (1995).
Initiation of reverse transcription of HIV-1 : secondary structure of the HIV-1 RNA}tRNA(3Lys) (template}primer). Journal of Molecular Biology 247, 236–250. Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B. & Kleiman, L. (1993). Identification of tRNAs incorporated into wild-type
and mutant human immunodeficiency virus type 1. Journal of Virology 67, 3246–3253. Levin, J. G. & Seidman, J. G. (1981). Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. Journal of Virology 38, 403–408. Li, X., Mak, J., Arts, E. J., Gu, Z., Kleiman, L., Wainberg, M. A. & Parniak, M. A. (1994). Effects of alterations of primer-binding site
sequences on human immunodeficiency virus type 1 replication. Journal of Virology 68, 6198–6206. Li, X., Quan, Y., Arts, E. J., Li, Z., Preston, B. D., De Rocquigny, H., Roques, B. P., Darlix, J.-L., Kleiman, L., Parniak, M. A. & Wainberg, M. A. (1996). Human immunodeficiency virus type 1 nucleocapsid
protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNALys3 in vitro : studies of viral RNA molecules mutated in regions that flank the primer binding site. Journal of Virology 70, 4996–5004. Mak, J., Jiang, M., Wainberg, M. A., Hammarskjold, M. L., Rekosh, D. & Kleiman, L. (1994). Role of Pr160gag-pol in mediating the selective
incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. Journal of Virology 68, 2065–2072.
IEA
Myers, G., Wain-Hobson, S., Henderson, L. E., Korber, B., Jeang, K. T. & Pavlakis, G. N. (1994). Human Retroviruses and AIDS 1994. A
Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, N. Mex., USA. Oude Essink, B. B., Das, A. T. & Berkhout, B. (1996). HIV-1 reverse transcriptase discriminates against non-self tRNA primers. Journal of Molecular Biology 264, 243–254. Peden, K. W. C. & Martin, M. A. (1996). HIV, a Practical Approach, pp. 21–45. Edited by J. Karn. Oxford : IRL Press. Peters, G. G. & Hu, J. (1980). Reverse transcriptase as the major determinant for selective packaging of tRNA’s into avian sarcoma virus particles. Journal of Virology 36, 692–700. Prats, A. C., Sarih, L., Gabus, C., Litvak, S., Keith, G. & Darlix, J. L. (1988). Small finger protein of avian and murine retroviruses has nucleic
acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO Journal 7, 1777–1783. Raba, M., Limburg, K., Burghagen, M., Katze, J. R., Simsek, M., Heckman, J. E., Rajbhandary, U. L. & Gross, H. J. (1979). Nucleotide
sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40transformed mouse fibroblasts. European Journal of Biochemistry 97, 305–318. Sprinzl, M., Hartmann, T., Weber, J., Blank, J. & Zeidler, R. (1989).
Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Research 17 (Suppl.). Varmus, H. & Swanstrom, R. (1984). RNA Tumor Viruses, 2nd edn, pp. 369–512. Edited by R. Weiss, N. Teich, H. Varmus & J. Coffin. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory. Vicenzi, E., Dimitrov, D. S., Engelman, A., Migone, T.-S., Purcell, D. F. J., Leonard, J., Englund, G. & Martin, M. A. (1994). An
integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. Journal of Virology 68, 7879–7890. Wakefield, J. K., Wolf, A. G. & Morrow, C. D. (1995). Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNALys3. Journal of Virology 69, 6021–6029.
Received 23 September 1996 ; Accepted 16 December 1996
SHORT COMMUNICATION
Sequence variation of the human immunodeficiency virus primer-binding site suggests the use of an alternative tRNALys molecule in reverse transcription Atze T. Das, Bep Klaver and Ben Berkhout Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3« end of this tRNA and a sequence near the 5« end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNALys3 as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNALys is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNALys5 and differs from tRNALys3 at five nucleotide positions. These results suggest that HIV uses both tRNALys3 and tRNALys5 molecules as primer, causing a switch of the PBS sequence.
The replication cycle of retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA, which is integrated into the host-cell genome (reviewed by Varmus & Swanstrom, 1984). This process is mediated by the virion-associated enzyme reverse transcriptase (RT) and a cellular tRNA is used as primer. This tRNA binds with its 3«terminal 18 nucleotides to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). Apart from the complementarity between the PBS and tRNA sequences, additional basepairing interactions between the tRNA primer and vRNA template may support this binding (Isel et al., 1995 ; Berkhout & Schoneveld, 1993 ; Aiyar et al., 1994). Furthermore, binding of the tRNA primer onto the Author for correspondence : Ben Berkhout Fax 31 20 6916531. e-mail B.Berkhout!AMC.UVA.NL
0001-4426 # 1997 SGM
vRNA can be activated by RT and nucleocapsid (NC) protein (Araya et al., 1979 ; Prats et al., 1988 ; Barat et al., 1989 ; Li et al., 1996). The RT protein, or the precursor Gag-Pol protein, was also suggested to be involved in selective encapsidation of the tRNA primer into virions (Peters & Hu, 1980 ; Levin & Seidman, 1981 ; Mak et al., 1994). The human immunodeficiency viruses (HIV-1 and HIV-2) and all simian immunodeficiency viruses efficiently replicate with tRNALys$ as primer. By mutating the PBS sequence, we (Das et al., 1995) and others (Li et al., 1994 ; Wakefield et al., 1995) recently demonstrated that HIV-1 can replicate with other tRNA primers (tRNAIle, tRNAHis, tRNALys",#, tRNAPhe, tRNAPro, tRNATrp), although less efficiently compared with the natural tRNALys$ primer. These mutants are unstable and revert to the wild-type PBSLys$ sequence upon prolonged culturing (Das et al., 1995). This reversion is mediated by annealing of a wild-type tRNALys$ onto the mutant PBS sequence, followed by copying of the 18 nucleotides at the 3« terminus of this tRNA primer during reverse transcription. During this mutation}reversion analysis, we occasionally observed a variant PBS sequence with a single C ! U substitution in the centre of the motif (Fig. 1). This suggests the presence of a variant tRNA primer resembling tRNALys$ in human cells. The PBS point mutation corresponds with a G ! A substitution at position 69 in the tRNA acceptor-stem (Fig. 2). A mouse gene encoding such a putative tRNALys species was previously described (Han & Harding, 1983). This murine tRNA contains five nucleotide differences compared with the human tRNALys$ sequence (positions 4, 15, 17, 48 and 69). We arbitrarily refer to this tRNA as tRNALys& (Fig. 2). The PBSLys& sequence was also observed in other mutation}reversion studies (Vicenzi et al., 1994 ; Das & Berkhout, 1995 ; A. T. Das and others, unpublished). Alignment of the PBS region of natural HIV-1 and HIV-2 isolates demonstrates considerable sequence variation up- and downstream of the PBS sequence, but relatively little variation is observed in the 18 nt PBS sequence (Fig. 3 ; Myers et al., 1994). Three out of the 24 sequenced HIV-2 isolates, but none of the 30 sequenced HIV-1 isolates, have a PBSLys& sequence. We tested the stability of the PBSLys& sequence in the HIV-2
IDH
A. T. Das, B. Klaver and B. Berkhout
Fig. 1. Primer-binding sites in HIV. PBS sequences (lower lines) complementary to tRNALys3 and tRNALys5 (upper lines) are found in HIV-1 and HIV-2 viruses, both natural isolates (Fig. 3) and viruses obtained in mutation/reversion studies. Nucleotides in the PBSLys5/tRNALys5 duplex that differ from the PBSLys3/tRNALys3 duplex are marked by a shaded box. Other PBS sequences that are not perfectly complementary to either tRNALys3 or tRNALys5 are not shown (n ¯ 2 for HIV-1 natural isolates, n ¯ 4 for HIV-2 natural isolates, n ¯ 3 for HIV-1 laboratory isolates). These variants may have resulted from PCR or sequencing errors. a, Combined results of studies in which the PBS site (Das & Berkhout, 1995 ; Das et al., 1995), the R-U5 region (A. T. Das and others, unpublished) and the U5 region (Vicenzi et al., 1994) were mutated.
Fig. 2. Secondary structure of tRNALys molecules. The cloverleaf structures of tRNALys5 (Han & Harding, 1983), tRNALys3, tRNALys1 and tRNALys2 are shown. tRNALys4 (not shown) is a tRNALys form specific for transformed cells that was initially thought to be an undermodified tRNALys2 (Raba et al., 1979), but turned out to be a differentially modified form of tRNALys2 (Hayenga et al., 1986). Nucleotides differing from tRNALys3 are shaded. Base modifications are indicated according to standard nomenclature (Sprinzl et al., 1989) ; A1, 1-methyladenosine ; A7, N-((9-β-D-ribofuranosylpurine-6-yl)carbamoyl)threonine ; A9, N-((9-β-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine ; C5, 5-methylcytidine ; D, dihydrouridine ; G2, N2-methylguanosine ; G7, 7-methylguanosine ; T3, 2«-O-methyl-5-methyluridine ; U9, 5-methoxycarbonylmethyl-2-thiouridine ; Ψ, pseudouridine.
isolate ROD (Peden & Martin, 1996). SupT1 T cells were transfected with the pROD10 proviral clone and cultured for 2 weeks as previously described (Das et al., 1995). The viruscontaining culture supernatant was used to infect fresh cells, which were cultured for another week. To determine the PBS sequence of the viral progeny, the LTR-leader region of the proviral DNA was amplified from total cellular DNA by PCR and cloned as described (Das et al., 1995). Four clones were sequenced and all contained a PBS sequence complementary to tRNALys$ instead of tRNALys&, indicating that the PBSLys& sequence is not stably maintained in the HIV-2 isolate. These combined results suggest that the variant tRNALys& molecule is used at a low frequency (approximately 3 %, Fig. 1) in reverse transcription by the HIV virus.
IDI
There are several possible explanations for the infrequent use of tRNALys& as primer in HIV replication. First and most importantly, this tRNA species may represent a so-called minor tRNA. Indeed, tRNALys& is not one of the three major tRNALys species identified in rabbit liver (tRNALys", tRNALys#, tRNALys$ ; Raba et al., 1979), and it is also not one of the four major tRNA species identified in HIV-1 virions (tRNALys", tRNALys#, tRNALys$, tRNAIle ; Jiang et al., 1993). Alternatively, inefficient packaging of tRNALys& may result in a low concentration of this tRNA species in virions, and thus in an infrequent priming with this tRNA during HIV replication. We note that the tRNALys" and tRNALys# variants, which have 15 and 13 base changes compared with tRNALys$, are packaged into HIV-1 virions (Jiang et al., 1993). Differences
tRNA primers in HIV replication
Fig. 3. PBS sequences of HIV-1 and HIV-2 isolates. The PBS sequences of HIV-1 and HIV-2 isolates are grouped according to the different subtypes (Myers et al., 1994). ^, deleted nucleotide ; y, insertion of ACTTGACGGTAATAGG (in the HIV-1 IBNG isolate). Three of the HIV-2 isolates have a deletion of one G-nucleotide in the PBS sequence. This may indicate that yet another tRNALys variant with a deletion of one C-nucleotide was used as primer for reverse transcription.
in PBS-annealing efficiency may also result in reduced tRNALys& usage. tRNALys$ and tRNALys& differ at five nucleotide positions (Fig. 2). The putative additional tRNAvRNA contacts involving the anticodon loop (Isel et al., 1995) will not be different for these two tRNA species, but one of the nucleotide changes (at position 69) affects the basepairing between the tRNA and the PBS (Fig. 1). At this position, tRNALys$ can form a G-C basepair with PBSLys$ and a G-U basepair with PBSLys&. In contrast, tRNALys& forms an A-U basepair with PBSLys& and an A-C mismatch with PBSLys$. Thus, annealing of tRNALys$ may be more efficient, resulting in the accumulation of PBSLys$ sites. Finally, the more frequent tRNALys$ usage may result from a more efficient priming by the HIV-1 RT enzyme. We note that this initiation reaction is rather specific because the related tRNALys" and tRNALys# primers are not efficiently used by the HIV-1 enzyme (Oude Essink et al., 1996). Consistent with this result, spontaneous PBSLys",# variation is never observed (Figs 1 and 3). Concluding, the occurrence of a PBSLys& sequence in HIV retroviruses suggests that a tRNALys& molecule is present in human cells. This minor tRNALys& species may play a specialized role in the metabolism of the host cell, but there is
currently no evidence for a specific role of this variant tRNA primer in HIV biology. We thank Wim van Est for artwork and Keith Peden for the gift of the HIV-1 pLAI and HIV-2 pROD10 proviral clones. This work was supported by the Dutch Cancer Society (KWF).
References Aiyar, A., Ge, Z. & Leis, J. (1994). A specific orientation of RNA secondary structures is required for initiation of reverse transcription. Journal of Virology 68, 611–618. Araya, A., Sarih, L. & Litvak, S. (1979). Reverse transcriptase mediated binding of primer tRNA to the viral genome. Nucleic Acids Research 6, 3831–3843. Barat, C., Lullien, V., Schatz, O., Keith, G., Mugeyre, M. T., Gru$ ningerLeitch, F., Barre! -Sinoussi, F., LeGrice, S. F. J. & Darlix, J. L. (1989).
HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO Journal 11, 3279–3285. Berkhout, B. & Schoneveld, I. (1993). Secondary structure of the HIV2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Research 21, 1171–1178.
IDJ
A. T. Das, B. Klaver and B. Berkhout Das, A. T. & Berkhout, B. (1995). Efficient extension of a misaligned
tRNA-primer during replication of the HIV-1 retrovirus. Nucleic Acids Research 23, 1319–1326. Das, A. T., Klaver, B. & Berkhout, B. (1995). Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). Journal of Virology 69, 3090–3097. Han, J. H. & Harding, J. D. (1983). Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis – nucleotide sequence of a mouse lysine tRNA gene. Nucleic Acids Research 11, 2053–2064. Hayenga, K., Hedgcoth, C., Harrison, M., Lin, V. K. & Ortwerth, B. J. (1986). Structural relationship between tRNA(Lys2) and tRNA(Lys4)
from mouse lymphoma cells. Molecular and Cellular Biochemistry 71, 25–30. Isel, C., Ehresmann, C., Keith, G., Ehresmann, B. & Marquet, R. (1995).
Initiation of reverse transcription of HIV-1 : secondary structure of the HIV-1 RNA}tRNA(3Lys) (template}primer). Journal of Molecular Biology 247, 236–250. Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B. & Kleiman, L. (1993). Identification of tRNAs incorporated into wild-type
and mutant human immunodeficiency virus type 1. Journal of Virology 67, 3246–3253. Levin, J. G. & Seidman, J. G. (1981). Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. Journal of Virology 38, 403–408. Li, X., Mak, J., Arts, E. J., Gu, Z., Kleiman, L., Wainberg, M. A. & Parniak, M. A. (1994). Effects of alterations of primer-binding site
sequences on human immunodeficiency virus type 1 replication. Journal of Virology 68, 6198–6206. Li, X., Quan, Y., Arts, E. J., Li, Z., Preston, B. D., De Rocquigny, H., Roques, B. P., Darlix, J.-L., Kleiman, L., Parniak, M. A. & Wainberg, M. A. (1996). Human immunodeficiency virus type 1 nucleocapsid
protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNALys3 in vitro : studies of viral RNA molecules mutated in regions that flank the primer binding site. Journal of Virology 70, 4996–5004. Mak, J., Jiang, M., Wainberg, M. A., Hammarskjold, M. L., Rekosh, D. & Kleiman, L. (1994). Role of Pr160gag-pol in mediating the selective
incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. Journal of Virology 68, 2065–2072.
IEA
Myers, G., Wain-Hobson, S., Henderson, L. E., Korber, B., Jeang, K. T. & Pavlakis, G. N. (1994). Human Retroviruses and AIDS 1994. A
Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, N. Mex., USA. Oude Essink, B. B., Das, A. T. & Berkhout, B. (1996). HIV-1 reverse transcriptase discriminates against non-self tRNA primers. Journal of Molecular Biology 264, 243–254. Peden, K. W. C. & Martin, M. A. (1996). HIV, a Practical Approach, pp. 21–45. Edited by J. Karn. Oxford : IRL Press. Peters, G. G. & Hu, J. (1980). Reverse transcriptase as the major determinant for selective packaging of tRNA’s into avian sarcoma virus particles. Journal of Virology 36, 692–700. Prats, A. C., Sarih, L., Gabus, C., Litvak, S., Keith, G. & Darlix, J. L. (1988). Small finger protein of avian and murine retroviruses has nucleic
acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO Journal 7, 1777–1783. Raba, M., Limburg, K., Burghagen, M., Katze, J. R., Simsek, M., Heckman, J. E., Rajbhandary, U. L. & Gross, H. J. (1979). Nucleotide
sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40transformed mouse fibroblasts. European Journal of Biochemistry 97, 305–318. Sprinzl, M., Hartmann, T., Weber, J., Blank, J. & Zeidler, R. (1989).
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Received 23 September 1996 ; Accepted 16 December 1996
