Institut Pasteur, Unité de Rétrovirologie Moléculaire, 28 rue du Dr Roux, 75724 Paris Cedex 15, France1
Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Klinikum Homburg, Haus 47, Universität des Saarlandes, 66421 Homburg/Saar, Germany2
Author for correspondence: Jean-Pierre Vartanian.Fax +33 1 45 68 88 74. e-mail jpvart{at}pasteur.fr
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Abstract |
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The HIV RT has several enzymatic activities requiring divalent cations as cofactors. For example, magnesium cations (Mg2+) are utilized for DNA polymerization, and Mg2+ and manganese cations (Mn2+) for ribonuclease H (RNase H) activity. The RNase H acts both as an endonuclease and an exonuclease. Both functions are catalysed by Mg2+, while RNase H-dependent hydrolysis of the double-stranded RNA intermediate is only possible in the presence of Mn2+ (Cirino et al., 1995 ). As far as DNA polymerase activity is concerned, substitution of Mg2+ by Mn2+ has been shown to increase the misincorporation of dNTPs and to alter the substrate specificities in vitro (Lazcano et al., 1992
; Valverde-Garduño et al., 1998
). This Mn2+-modulated enzyme function is not unique to HIV RT. It has also been reported for avian myeloblastosis virus (AMV) RT (Sirover & Loeb, 1977
), Moloney murine leukaemia virus RT (Gerard & Grandgenett, 1975
; Van Beveren & Goulian, 1979
), T4 DNA polymerase (Goodman et al., 1983
), Taq DNA polymerase (Cadwell & Joyce, 1992
; Fromant et al., 1995
; Leung et al., 1989
; Vartanian et al., 1996
), E. coli polymerase I (Richetti & Buc, 1993
) and human DNA polymerases
and ß (Chang & Bollum, 1973
).
The aim of this work was to study the effect of Mn2+ on the mutation rate following HIV replication in culture (ex vivo). Briefly, 3x106 cells of the monocytic cell line U937-2 were preincubated with 0·5 mM MnCl2 in RPMI 1640 medium for 30 min at 37 °C. This medium contains 0·4 mM Mg2+ but lacks Mn2+. The cell culture was then infected with 6x102 c.p.m. of HIV-1 Lai RT activity per 106 cells as described (Nietfeld et al., 1995 ). After 3 h at 37 °C, cells were washed twice with RPMI 1640 medium and then cultured in RPMI 164010% foetal calf serum with 0·5 mM MnCl2 for 16 h. Control cell cultures without MnCl2 treatment were infected accordingly. The cells were then washed, centrifuged and resuspended in 1 ml PBS (without Ca/Mg) containing 1 µg/ml DNase I and 10 mM MgCl2. After 30 min at room temperature, the cells were centrifuged and resuspended in 200 µl 2x lysis buffer for PCR containing 10 mM Tris pH 8·3, 1% Tween, 1% NP40 and 0·6 mg/ml proteinase K. Samples were maintained for 1 h at 55 °C. Proteinase K was inactivated by incubating for 15 min at 95 °C and the samples were kept at -20 °C until use. A fragment of the HIV RT region [codons 550557 from the start of the pol open reading frame (Wain-Hobson et al., 1985
)] was amplified by PCR with the primers RTGA1 (5' GGCGAATTCTAAATTTAAACTACCCATACAA) and RTGA2 [5' GGCAAGCTTGTGG(C/T)TTGCCAATA(C/T)T(C/T)TGT] (Fig. 1
). Amplification products were cloned in-frame into the lacZ
-coding fragment of M13mp18 via EcoRI and HindIII. E. coli XL-1 Blue was transformed and plated on 8% X-Gal plus IPTG indicator plates. Colonies obtained were counted and all white plaques were sequenced as previously described (Vartanian et al., 1997
).
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Manganese treatment of permissive cells increased significantly the error rate for HIV reverse transcription ex vivo by more than 6-fold. Five mutants out of 980 clones were identified in the Mn2+-treated sample whereas no mutant within 1359 clones was found in the control (Table 1). Sequencing of the individual mutants identified seven point mutations, four G
A transitions within the tryptophan codons and three additional transversions in the glutamic acid and threonine codons leading to aspartic acid and serine respectively (Fig. 1). From these data, the minimal HIV mutation frequency (no. of mutations/no. of sequence nucleotides multiplied by the no. of total clones analysed) during Mn2+ treatment can be calculated to be 3x10-4. This is only a minimal estimate because mutations that do not give rise to an altered ß-galactosidases activity or to stop codons are missed. Therefore, compared to the control infection with a mutation frequency <3x10-5 (at least one white colony per 1359 blue), manganese treatment increased the mutation frequency at least by a factor of 10. As the experiments lasted 16 h, the frequencies correspond to a single HIV replication round.
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The chemical basis by which Mn2+ induces an aberrant polymerization activity of the HIV RT is not completely understood. Mn2+ seems to decrease the number of RT molecules engaged in polymerization (Valverde-Garduño et al., 1998 ). This cation is known to reduce base stacking by binding to the primertemplate helix (Shin, 1973
; Vamvakopoulos et al., 1977
). This could alter the stability of the nucleic acid molecule and confer more flexibility to the helix allowing an easier introduction of misincorporated nucleotides (Sala et al., 1995
). On the other hand, alterations in helix structure in direct contact with the enzyme could induce an increase in the enzyme's koff, thus facilitating RT to fall off the primertemplate complex. In addition, Mn2+ is known to bind to the heterocyclic nitrogen atoms of bases and to stabilize mismatches which expose heterocyclic nitrogen atoms to the solvent with a higher efficiency than Mg2+ (Pan et al., 1993
). In this way Mn2+ could increase the probability of fixing mutations in the HIV genome. Furthermore, Mn2+ could also bind noncovalently at the metal-binding sites in the RT structure. Altogether, the effect of Mn2+ on the RT polymerization activity seems to be multifactorial, involving simultaneous interactions with the template, the substrate and the enzyme.
In conclusion, the fidelity of the HIV RT can be manipulated by various means with subsequent mutation frequencies spanning from the basal 3·4x10-5 ex vivo (Mansky & Temin, 1995 ) to 10-2 in vitro (Martinez et al., 1995
; Sala et al., 1995
; Vartanian et al., 1997
). Knowing how to reduce RT fidelity especially ex vivo might offer tools to directly increase the HIV mutation rate. While HIV is able to tolerate a high degree of sequence divergence without changes in pathogenicity, it might be possible to increase the mutation rate beyond the error threshold (Domingo & Holland, 1997
). This would have to be achieved using small synthetic molecules for manganese and biased dNTP concentrations would probably prove mutagenic for host cell replication. However, the extensive collection of RT structures might allow modelling of a molecule that could relocate the nucleic acid helix in the cleft so altering fidelity.
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References |
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Received 12 February 1999;
accepted 28 April 1999.