1 Centro Nacional de Microbiología (CNM), Instituto de Salud Carlos III, Majadahonda, Madrid 28220, Spain
2 Centro Sanitario Sandoval (CSS), Comunidad Autónoma de Madrid, Madrid 28010, Spain
Correspondence
Cecilio López-Galíndez
clopez{at}isciii.es
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ABSTRACT |
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INTRODUCTION |
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In HIV-1 quasispecies, the presence of highly divergent nucleotide sequences within quasispecies has been detected in sequential samples from the same patient (Delwart et al., 1997; Halapi et al., 1997
; Wolinsky et al., 1996
), and also from different tissue compartments of the same patient (Wong et al., 1997b
; Delwart et al., 1998
; Itescu et al., 1994
; Poss et al., 1998
). Highly divergent variants have been reported in single blood samples generally associated with double infections (Diaz et al., 1995
; Sala et al., 1994
; Zhu et al., 1995
). In this study, we analysed HIV-1 quasispecies from three Spanish patients infected for more than 9 years and displaying divergent nucleotide sequences without evidence of double infection. We determined the origin of these nucleotide sequences by phylogenetic analysis and by dating the origin of the different clades forming the quasispecies. Nucleotide sequence groups within individual quasispecies had dates of origin up to 10 years apart. Some clades were considered ancestral because they dated close to the seroconversion time and they displayed a short genetic distance to the node of the patient's HIV-1 phylogenetic tree.
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METHODS |
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Amplification and quantification of viral nucleic acids.
PCR and RT-PCR amplification conditions were as described by Casado et al. (2001). Proviral load was carefully determined as previously described (Rodrigo et al., 1997
) in the same nested PCR as used to amplify the entire gp120. To avoid genetic bottlenecking, the first PCR included at least 20 copies of proviral DNA and primers 91ECU (5'-CTTAGGCATCTCCTATGGC-3', 59565974 HXB2 position) and 22ED (described by Casado et al., 2001
) were used. The second PCR included 1 µl of the first PCR products and primers 27EU (described by Casado et al., 2001
) and 96ED (5'-AGACAATAATTGTCTGGCCTGTACCGT-3', 78627836 HXB2 position). Plasma HIV-1 RNA was quantified with the Amplicor HIV Monitor test kit with a detection limit of 50 copies ml-1 (Roche Diagnostics Systems), following the manufacturer's instructions. Nested RT-PCR was used to amplify the V3 env region. The first RT-PCR included at least 100 copies of viral RNA and primers 169ECU (5'-AATGGCAGCACAGTACAATGTACAC-3', 69456969 HXB2 position) and 96ED were used. The second PCR included 1 µl of the first RT-PCR products and primers 27EU and 28ED (5'-ATGAATTCTGGGTCCCCTCCTGAGGA-3', 73147339 HXB2 position).
Cloning and sequencing.
Two microlitres of the nested PCR products were ligated into plasmid pCR 2.1 and cloned according to the TA Cloning Kit instructions (Invitrogen). Eighteen to twenty clones per sample were sequenced with primer 27EU using the ABI PRISM Dye Terminator reaction kit (Perkin-Elmer) according to manufacturer's instructions in an ABI PRISM 377 automated sequencer (Perkin-Elmer).
Phylogenetic analysis.
All nucleotide sequence analyses were carried out on a 614 bp fragment of the env gene spanning from the distal portion of C2 to the proximal portion of C5 (nucleotides 70687682 in the HXB2 clone). Nucleotide sequences were aligned using CLUSTAL W (Thompson et al., 1994) and later hand edited. All positions with an alignment gap in at least one nucleotide sequence were excluded from the analysis, giving a 551 nucleotide fragment. All tree reconstructions in the study were performed by two independent methods: the neighbour-joining (NJ) method (with Kimura two-parameter model and a transition/transversion ratio of 2·0) in 1000 bootstrapped datasets as implemented in the MEGA version 2.1 program (Kumar et al., 2001
); and by the maximum-likelihood (ML) method with the DNAML program (random input order, empirically found base frequencies and transition/transversion ratios and single rate of substitution) as implemented in PHYLIP version 3.6 (Felsenstein, 1993
). The likelihood of the DNAML tree was significantly higher than that of the DNAMK tree (which assumes that all sequences are equidistant to the node of the tree) with a log likelihood ratio test (P<0·001).
Nucleotide distance analysis.
Intrasample (heterogeneity) and intersample (divergence) genetic distances were estimated by the Kimura two-parameter model (as indicated above) using MEGA version 2.1 (Kumar et al., 2001) in 100 bootstrapped datasets and by the F84 model (random input order, empirically found base frequencies and transition/transversion ratios and single rate of substitution) using the DNADIST program as implemented in PHYLIP version 3.6. Heterogeneity for patients 10, 30 and 45, and divergence for patient 30 clade a, were expressed as the mean distance for all pairwise comparisons between nucleotide sequences within a sample or from two different samples, respectively. Divergence for control patients C1 and C2 was calculated as the genetic distance between the global nucleotide sequences in two samples. The most recent common ancestor (MRCA) for each patient was inferred with the DNAML program (PHYLIP version 3.6) (Felsenstein, 1993
). This sequence represents the most distal node of the tree originating all HIV-1 nucleotide sequences from the patient. Mean genetic distances between nucleotide sequence clusters and each patient MRCA were estimated by the F84 model (as indicated above) using DNADIST (PHYLIP version 3.6) (Felsenstein, 1993
).
Dating of viral nucleotide sequences within quasispecies.
HIV-1 subtype B V3 env nucleotide sequences from 96 Spanish samples, collected from 1993 to 2002, were generated by us from homosexual and injecting drug users from the Centro Sanitario Sandoval (CAM, Madrid) and from the Hospital General de Navarra (Casado et al., 2000). A consensus nucleotide sequence (Spanish consensus), which included the most frequent nucleotide in each position, was generated as described by Casado et al. (2000)
. The MRCA for this nucleotide sequence set (Spanish MRCA) was also inferred by the DNAML program as explained above. Samples were grouped by the collection year, and the genetic distance of each sample to the Spanish consensus or to the Spanish MRCA was estimated by the Kimura two-parameter model and by the ML method, respectively, with the DNADIST program using the conditions described above. For dating of the patient's clades, the mean genetic distance of all pairwise comparisons between nucleotide sequences from each cluster to the Spanish consensus or to the Spanish MRCA was used (see Fig. 2
).
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RESULTS |
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Temporal evolution of proviral quasispecies
To further analyse the presence and temporal evolution of the different nucleotide sequence clades in the proviral quasispecies, the same viral population analysis was carried in consecutive samples of the two low viral load patients; the results are shown for patient 30 in Fig. 3. In the 1998 sample the quasispecies was dominated by the ancestral clade c and the modern clade b sequences, representing 52 % and 35 % respectively. In the following years there was a rapid turnover of the proviral population and the quasispecies was dominated by sequences of the recent clade a, although the recent b population showed a slower turnover than the ancestral c clade. Patient 45 showed a similar pattern of temporal evolution, with the detection of ancestral nucleotide sequences in the first two samples and their replacement by the recent ones in later samples (data not shown).
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DISCUSSION |
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The ancestral nucleotide sequences were defined by two criteria: first, by their short distance to the MRCA of each patient; and second, by the early date of the sequences reflected in the short distance to the Spanish consensus or Spanish MRCA sequence. The correlation between genetic divergence and time was used by our group (Casado et al., 2000) and by Lukashov & Goudsmit (2002)
for the dating of the HIV-1 epidemic in Spain and the USA and the Netherlands respectively. Furthermore, a correlation between genetic divergence and time was also used for the dating of an individual sequence from an old HIV-1 sample (Zhu et al., 1998
). Genetic distance to patient MRCA was also used to characterize the presence of ancestral sequences in treated patients (Gunthard et al., 1999
). These analyses were based upon the existence of a molecular clock in in vivo HIV-1 evolution, which was demonstrated by Leitner & Albert (1999)
, using the same V3 region, and by Lukashov & Goudsmit (1997)
. There are controversies regarding the use of genetic distances for the dating of individual samples because of the wide dispersion of the genetic distance with time due to the heterogeneous nature of the HIV-1 infection (Korber et al., 1998
; Wolinsky et al., 1996
; Zhu et al., 1998
). However, the genetic distance dispersion is bigger when comparisons are made between subtypes (Korber et al., 1999
), but our analysis was performed in the context of an epidemic caused by the introduction and circulation of a single variant of subtype B in Spain (Casado et al., 2000
). Finally, although every dating approach has an error and it is conditioned by the estimation used, the differences in the dates of the clades between 7 and 11 years were greater than any calculation error.
The old date and small genetic distance to patient's MRCA of the ancestral nucleotide sequences is incompatible with continuous virus replication for 910 years because divergence values for HIV-1 have been estimated at around 1 % per year and then could only be the result of viral latency (Shankarappa et al., 1999). In addition, the detection of these ancestral sequences at high proportion 10 years after their date of origin suggest a recent activation and/or a clonal expansion of the memory or naïve CD4+ T cell latent reservoir (Cheynier et al., 1998
), but not the long-term persistence of ancestral sequences (Simmonds et al., 1991
) because of the lack of intermediate sequences. The best evidence that the ancestral nucleotide sequences in the proviral population resulted in active replicating virus was the discovery, in the 2002 sample of patient 30, of one recombinant clone displaying ancestral and recent sequences. In our patients, however, the presence of the ancestral nucleotide sequences in the proviral viral populations was not correlated with their presence in the plasma viral populations. Plasma RNA viral quasispecies of patients 10 and 30 were analysed, but no RNA ancestral nucleotide sequences were detected, following the procedure described in Methods. This could be explained by the faster turnover of the viral RNA populations compared with the proviral ones (Simmonds et al., 1991
), and/or by the lower fitness of early variants in comparison to late ones because of the continuous optimization associated with virus replication (Nijhuis et al., 1999
). A similar result was observed in a study by Bagnarelli et al. (1999)
, where 12 out of 36 proviral clones were ancestral in one patient, while only one archival clone out of 44 was detected in the plasma.
The persistence of ancestral sequences could have important implications for the understanding of the in vivo evolution of HIV-1. First, the follow-up of patient 30 showed that changes in the proportion of the ancestral and recent clusters in the quasispecies have profound effects in the mean values of divergence, diversity and dN/dS (data not shown) and could result in discontinuities in the pattern of viral evolution as observed in other studies (Ostrowski et al., 1998; Simmonds et al., 1991
). Second, the activation of HIV-1 variants resident in memory or naïve T-cells illustrates the importance of stochastic processes in HIV-1 population dynamics (Cheynier et al., 1994
, 1998
), although it does not rule out the impact of immune selective forces (Delwart et al., 1997
; Lukashov et al., 1995
; Wolinsky et al., 1996
). In fact, while immune selection seems to drive evolution of clusters a and b, a stochastic activation probably conditioned the appearance of cluster c.
It is of great interest to know if the persistence of ancestral nucleotide sequences is a general phenomenon in HIV-1 infection. In the present study, the three patients analysed should be considered as slow progressors (SP) (see Table 2). A cluster of archival nucleotide sequences was also detected in another SP patient (Ostrowski et al., 1998
) and in three Italian SP patients (Bagnarelli et al., 1999
). Nevertheless, the detection of the persistence and activation of wild-type viruses after many years under antiretroviral therapy in HIV-1-infected patients (Finzi et al., 1997
; Wong et al., 1997a
), and the detection of ancestral nucleotide sequences in SIV infection (Ryzhova et al., 2002
), suggest the generality of this phenomenon.
This work has shown the co-existence of recent and ancestral sequences within viral quasispecies in HIV-1-infected patients, and confirmed that a population of latently infected cells harbouring ancestral nucleotide sequences could persist despite many years (up to 10) of ongoing virus replication in untreated HIV-1 patients. The analysis of the causes and the frequency of ancestral viral variants will be helpful not only for the understanding of the evolutionary dynamics of HIV-1 in vivo but also for the pathogenesis and treatment of HIV-1 infection.
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ACKNOWLEDGEMENTS |
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Received 22 May 2003;
accepted 26 September 2003.