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Journal of Virology, August 2001, p. 7184-7187, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7184-7187.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Efficacy of Dideoxynucleosides against Human Foamy
Virus and Relationship to Its Reverse Transcriptase Amino Acid
Sequence and Structure
Anne
Yvon-Groussin,1
Pierre
Mugnier,1
Philippe
Bertin,2
Marc
Grandadam,1
Henri
Agut,1
Jean Marie
Huraux,1 and
Vincent
Calvez1,*
Department of Virology, UPRES EA 2387,
Pitié-Salpêtrière Hospital,1
and Department of Biochemistry and Molecular Genetics,
Institut Pasteur,2 Paris, France
Received 17 April 2000/Accepted 23 April 2001
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ABSTRACT |
Human foamy virus (HFV), a retrovirus of simian origin which
occasionally infects humans, is the basis of retroviral vectors in
development for gene therapy. Clinical considerations of how to treat
patients developing an uncontrolled infection by either HFV or
HFV-based vectors need to be raised. We determined the susceptibility
of the HFV to dideoxynucleosides and found that only zidovudine was
equally efficient against the replication of human immunodeficiency
virus type 1 (HIV-1) and HFV. By contrast, zalcitabine (ddC),
lamivudine (3TC), stavudine (d4T), and didanosine (ddI) were 3-, 3-, 30-, and 46-fold less efficient against HFV than against HIV-1,
respectively. Some amino acid residues known to be involved in HIV-1
resistance to ddC, 3TC, d4T, and ddI were found at homologous positions
of HFV reverse transcriptase (RT). These critical amino acids are
located at the same positions in the three-dimensional structure of
HIV-1 and HFV RT, suggesting that both enzymes share common patterns of inhibition.
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TEXT |
Spumaviruses, in addition to
oncornaviruses and lentiviruses, constitute the third genus of the
Retroviridae family and are known to be widely prevalent in
primates (15). The first spumavirus isolate found
in humans was obtained in 1971 from a patient with a nasopharingeal
carcinoma (1). This isolate was named human foamy
virus (HFV). The sequence data of foamy virus isolates from chimpanzees
support the hypothesis that HFV is not a human prototype but rather a
variant strain of a simian foamy virus (21). A substantial
seroprevalence (1.8%) of infection with simian foamy virus
among humans occasionally exposed to nonhuman primates was also
described. These infections have not as yet resulted in either disease
or sexual transmission and might represent benign endpoint infections
(7). Due to its apparent lack of pathogenicity and its
capacity to induce proviral genome integration in nondividing cells,
HFV is used to generate retroviral vectors in development for gene
therapy (8, 19, 27). Clinical considerations of how to
treat patients developing an uncontrolled infection by either HFV or
HFV-based vectors need to be raised. The nucleotide sequence of HFV was
determined and showed that the pol gene was divided into
three domains: reverse transcriptase (RT), RNase H, and integrase
(9, 14, 17). Biological properties of the
corresponding HFV pol gene products were determined
(9). The RT of HFV might be an attractive target for
efficient antiviral chemotherapy, due to the known inhibitory effect of
nucleoside analogs on human immunodeficiency virus type 1 (HIV-1) RT
and their well-demonstrated activity against HIV-1 infections in vivo. In this study, we analyzed the relationship between the activity of RT
dideoxynucleoside inhibitors against HFV, the RT amino acid sequence of
HFV RT, and its three-dimensional (3D) structure.
Susceptibility of HFV to dideoxynucleosides.
Virus stocks were
obtained by infecting U373MG cells (American Type Culture Collection,
Manassas, Va.) with the HFV prototype strain (kindly provided by G. Peries, Saint Louis Hospital, Paris, France) for 10 days until an 80%
cytopathic effect (CPE) was observed (1). Five HIV-1 RT
inhibitors were tested: azidothymidine (zidovudine [ZDV]; Sigma),
dideoxyinosine (didanosine [ddI]; Sigma), dideoxycytidine (zalcitabine [ddC]; Sigma), lamivudine (3TC; Glaxo Wellcome) and stavudine (d4T; Sigma). The susceptibility of HFV to these drugs was
determined by classical procedure: 2 × 104
cells per well were seeded in 96-well plates to obtain a subconfluent cell monolayer after culture for 24 h. Cells were infected in quadruplicate with 50 µl of fivefold serial dilutions of virus stock
per well. Antiviral drugs were added at the same time. The antiviral
drug concentrations were those usually used for HIV-1 (4)
and scaled up when 50% inhibitory concentrations
(IC50s) (i.e., the concentration reducing viral
infectious titers by 50%) were out of range. After 10 days,
plates were checked for the presence of a CPE in each well. The
infectious titers in the absence and presence of drugs were calculated
from the number of wells exhibiting a CPE at each concentration by the
Reed and Münch method (4). All four experiments were
done with two stocks of HFV. The susceptibility profile of HFV to an
antiretroviral drug was expressed as the IC50.
The IC50s for HFV were compared to published
IC50s for wild-type-sensitive HIV-1
(4) (Table 1). ZDV appeared
to be the most effective drug against HFV, and its
IC50 was identical to that published for HIV-1.
By contrast, IC50s of ddC, 3TC, d4T, and ddI were
3-, 3-, 30-, and 46-fold higher, respectively, than that for HIV-1,
reflecting a lower sensitivity of HFV to these drugs.
HFV sequence analysis and comparison to HIV-1 RT amino acids.
We sequenced the RT active-site region of our HFV strain. The region
was amplified by PCR on proviral DNA with primers RT1 (5'-CTGGTGATTATCCTCCTCGCCC-3') and RT2
(5'-AAAAGTGTCTGTTAGGCCACGACC-3') (40 cycles, each cycle at
94°C for 1 min, 62°C for 1 min, and 72°C for 1 min). A
fragment of 652 bp was obtained and sequenced with an automated DNA
sequencer (ABI; Perkin-Elmer). Sequence analysis with Geneworks
software revealed no difference between our strain and the previously
published sequence (14). To locate the homologous amino
acid residues of HFV and HIV-1 RT, we performed an amino acid alignment
of the deduced HFV and HIV-1 sequences with the Multalign
algorithm (3), refined manually (data not shown), which
confirmed those sequences previously published (14). The
similarity between these proteins is low (about 20% identical amino
acid residues between the two proteins). However, this limited homology
extends over the entire length of both proteins, and several conserved
features were revealed by the alignment. In particular, the presence of
strictly conserved residues in all RNA-dependent polymerases
characterized so far (18) was observed. This permitted us
to investigate the relationship between the spectrum of susceptibility
to dideoxynucleosides and the amino acid residues present at crucial
homologous positions (Table 2). Some of
the classical amino acid mutations which have been involved in the
HIV-1 resistance to dideoxynucleosides are naturally present in HFV RT
amino acid sequences, at the HIV-1 positions 74 and 184 for ddI and ddC
(5, 6, 24), position 184 for 3TC (20, 26) and
position 75 for d4T (11, 13; D. Shepp, A. Ashraf, C. Millian, and R. Pergolizzi, Abstr. 2nd Natl. Conf. Human Retrovir., abstr. 139, 1995).
At other positions, HFV amino acids were different from those present
in sensitive HIV-1 strains but also distinct from those observed in
resistant HIV-1 strains. In the case of ZDV, which exhibited the same
activity level on HFV and HIV-1, we did not find any of the amino acid
residues involved in HIV-1 resistance to the drug at positions 41, 67, 70, 215, and 219 (12), corresponding to positions 91, 118, 121, 252, and 256 of the HFV RT. In addition, neither multidrug
resistance mutations (Q151 M profile) (22, 23) nor
a position 69 insertion (J. J. de Jong, S. Jurriaans, J. Goudsmit,
E. Baan, R. Huismans, S. A. Danner, M. E. Hillebrand, J. H. ten Veen, and F. de Wolf, Antivir. Ther., abstr. 18, 1998) of
resistant HIV-1 strains was observed in the case of HFV. These results
are in accordance to the susceptibility pattern of HFV to
dideoxynucleosides depicted in Table 1.
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TABLE 2.
Amino acid residues of HIV-1 RT involved in resistance to
dideoxynucleosides and correspondence to amino acid residues of HFV
RT
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HFV RT 3D structural modeling.
We determined a putative 3D
structural modeling of HFV RT to check if the crucial positions defined
by sequence alignment were located at the same 3D positions as in HIV-1
RT. The molecular modeling of HFV was calculated by homology with the
3D structure of HIV-1 (BH10 isolate) RT (3HVT.pdb) with the Swiss Model Automated Protein Modelling service at the Glaxo Wellcome Experimental Research Center (Geneva, Switzerland), which makes use of the ProMod
software (16). Secondary structure and distribution of hydrophobic clusters were analyzed as previously described
(2), and the results suggested that the structural
organization of both proteins was similar (data not shown). Therefore,
on the basis of the alignment, we calculated the 3D structure of the HFV RT by homology with the 3D structure of HIV-1 enzyme, which has
been determined by X-ray diffraction (10). This model
markedly suggested that the structural topology was conserved in both
proteins (Fig. 1), in particular, the
organization in different subdomains (fingers, palm, thumb, and
connection regions) (25). Moreover, the amino acids
corresponding to the catalytic triad of aspartic acids (Asp 110, Asp
185, and Asp 186 for HIV-1 and Asp 161, Asp 223, and Asp 224 for HFV),
as well as those related to dideoxynucleoside resistance (Asp 74, Asp
75, and Asp 184 for HIV-1 and Asp 124, Asp 125, and Asp 222 for HFV),
are located at similar positions in the 3D structure of both proteins.
This 3D representation permitted us to verify that the amino acids in
HFV RT corresponding to those involved in HIV-1 dideoxynucleoside
resistance were located at the same 3D positions. The fact that
critical amino acid residues involved in resistance are located in the
same subdomains in both proteins confirms the previous results
identifying the exact localization of HFV RT gene (9).
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FIG. 1.
Predicted 3D structure of HFV RT based on the known
structure of HIV-1 RT.  -Helices are indicated in red, and
 -strands are indicated in yellow. Essential aspartates (Asp 110, Asp
185, and Asp 186 for HIV-1 and Asp 161, Asp 223, and Asp 224 for HFV),
which may bind the triphosphate moiety of incoming nucleotide analogs
and deoxynucleoside triphosphate substrates via Mg2+, are
indicated as orange spheres. White spheres indicate two positions
involved in resistance of HIV-1 to dideoxynucleoside (amino acids 74 and 184) and the homologous position in HFV RT.
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Our work studied the efficacy and the relationship between
dideoxynucleoside analog susceptibility, amino acid sequence, and
structure of HFV RT. We evaluated the HFV susceptibility profile
of
five dideoxynucleoside analogs commonly used in HIV-1 treatment.
The
IC
50 values were shown to be higher than those
previously
described for HIV-1, except that of ZDV. These results are
in
accordance with those recently published (
28). To
correlate
function inhibition and structure, we determined the RT amino
acid sequence of the HFV strain tested, and we performed alignment
with
HIV-1 RT. The genomic determinants of the natural resistance
pattern of
HFV to the dideoxynucleosides tested were found to
be homologous to
those previously described for HIV-1 resistance,
despite a low
similarity between HFV and HIV-1 RT. A 3D model
of HFV RT confirmed
these results, which might have important
implications in both the
studies of retrovirus phylogeny and the
management of HFV infections in
vivo.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology, CERVI, Pitié-Salpêtrière Hospital, 47-83
Bd. de l'Hôpital, 75651 Paris Cedex 13, France. Phone: 33 1 42 17 74 01. Fax: 33 1 42 17 74 11. E-mail:
vincent.calvez{at}psl.ap-hop-paris.fr.
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Journal of Virology, August 2001, p. 7184-7187, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7184-7187.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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