Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France
Correspondence
Rémi Cheynier
remi{at}pasteur.fr
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ABSTRACT |
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Present address: CNRS/UNSA UMR 6097, IPMC, 660 Route des Lucioles, 06560 Valbonne, France.
Present address: Unité des Virus Lents, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
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MAIN TEXT |
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Lymphoid-tissue CD4+ T lymphocytes are the major producers of virus (Stahl-Hennig et al., 1999; Tenner-Racz et al., 1998
; Veazey et al., 1998
; Zhang et al., 1999
). Moreover, a greater proportion of CD4 T cells harbour provirus than interdigitating dendritic cells or macrophages (McIlroy et al., 1995
, 1996
). Viral proliferation is sustained in the immunocompetent structures of secondary lymphoid organs (i.e. GC and T-cell zones in lymph nodes, GCs and periarteriolar lymphoid sheath in the spleen) as a consequence of antigen-specific CD4+ T-cell activation (Cheynier et al., 1998
; Ostrowski et al., 1998
). Activation-dependent viral proliferation leads to local development of viral quasispecies in GCs, such that a unique viral population characterizes each splenic white pulp (Cheynier et al., 1994
; Gratton et al., 2000
).
The origin of viruses in GCs is unknown. They are not produced by FDCs, as this cell type is not permissive for HIV infection (Embretson et al., 1993; Grouard & Clark, 1997
; Reinhart et al., 1997
; Schmitz et al., 1994
; Tsunoda et al., 1996
). Two hypotheses present themselves: virus, mainly in the form of circulating immune complexes, may be deposited on the FDC surfaces; this hypothesis has some support from murine experiments (Heath et al., 1995
). This may be referred to as a peripheral origin. Another possibility is that virus may be produced locally by HIV-infected antigen-specific T cells within GCs and deposited on the FDC surfaces. This may be termed a local origin.
Although the two hypotheses are not mutually exclusive, how might it be possible in a natural setting to distinguish between the two, or, more precisely, to determine which is the more important? The enormous genetic variation of HIV-1 needs no reminder. More to the point, it varies considerably in space (Delassus et al., 1992), so much so that different splenic white pulps (Cheynier et al., 1994
) and even 10 µm sections through splenic GCs (Gratton et al., 2000
) exhibit different sequences. This suggests an experimental approach to distinguishing the above alternative.
If the majority of virus present within the white pulps is of peripheral origin, the sequence of genomic RNAs from different white pulps should be generally similar. In that case, one should expect that DNA- and RNA-derived sequences from the same white pulps will be different, as the infiltrating cells might have been infected earlier during the course of disease development. If, on the other hand, virus is derived mainly from infected T cells within the white pulp, then different sequence sets should characterize HIV-1 virions between two white pulps. Equally, some degree of correspondence should exist between viral DNA-, genomic RNA- and spliced mRNA-derived sequences.
Spleens of three HIV-1 patients who underwent surgery for non-treatable thrombocytopenia or suspicion of Burkitt's lymphoma were studied. Their peripheral blood CD4+ T-cell counts were all >200 µl1. Immediately after surgery, spleens were cut into pieces (23 cm3) and splenic white pulps were dissected under a binocular microscope (magnification x10).
The first spleen analysed was from patient B, who was not on therapy at the time of splenectomy (5900 RNA copies ml1, 583 CD4 µl1). Total DNA and RNA were extracted independently from three white pulps by using a Master Pure extraction kit (Epicentre). To avoid contamination, PCR was performed in a DNA-free room. In order to amplify viral DNA sequences, PCR for the V1V2 hypervariable env region was performed by using the LV15/LV13 and SK122/123 primer pairs (Cheng et al., 1994; Delassus et al., 1992
) on DNA extracts. For viral and mRNA amplifications, cDNA synthesis was performed on RNA extracts by using the LV13 primer. The LV15 primer served as 5' primer for full genomic RNA, whilst an outer 5' primer, LTR-SD (5'-TCTCTCGACGCAGGACTCGGCTTG-3'), mapping 42 bp 5' to the major splice leader for HIV-1 Lai, was used for spliced env mRNA. For the nested reaction, SK122/123 primers were used. Accordingly, RT-PCR amplification using oligonucleotides specific for spliced mRNA detects intracellular spliced mRNA specifically. To control for the absence of residual DNA in RNA preparations, PCR was performed on purified RNA (without the RT step), using env V1V2-specific primers. No preparations proved positive.
For each white pulp, PCR products were cloned in M13mp18; approximately 20 sequences were established from DNA, total RNA and env mRNA and are given in the form of an unrooted phylogenetic tree (Fig. 1), compiled by using the SplitsTree2 program (Bandelt & Dress, 1992
; http://bibiserv.techfak.uni-bielefeld.de/splits/). The majority of sequences were clustered into three groups based on their spatial origin. A predominant form was invariably accompanied by a collection of variants. Whether they were derived from DNA, total RNA or env mRNA, the major forms were identical or very similar to one another. For two white pulps, B2 and B3, the predominant DNA sequences (D) were identical to that derived from genomic RNA (R). For the third white pulp, B1, there were two point mutations between the predominant forms. A comparable situation pertained to the DNA and spliced mRNA (Rs) collections of sequences. Only for B2 was there complete coincidence of the predominant DNA, RNA and mRNA forms. The difference between two sequences from the same white pulp was always smaller than the distances between the three white pulps (Fig. 1
). A small number of sequences did not conform to this distribution based on lymphoid architecture. For example, one of 40 sequences from B2, four of 53 from B1 and four of 51 from B3 mapped in a spatially discordant manner. Hence, it may be concluded that the majority (>90 %) of genomic RNA is produced in situ.
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The sequences could be resolved into five distinct clades, differing from each other by a minimum of 18 mutations (Fig. 2). In white pulp C19, there was close concordance between extracellular viral RNA-derived and cell (proviral or non-integrated viral DNA)-derived sequences for clusters A and B. Other sequences were derived from either DNA or RNA fractions (clades C and D). For white pulp C18, both RNA- and DNA-derived sequences were essentially clustered into two clades (D and E) with only two of 30 (6·6 %) being scattered across the other three clades.
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FDCs are unique to lymphoid follicles of secondary lymphoid organs. They are of non-haematopoietic origin and play a critical role in the maturation of immunoglobulin-producing cells and establishment of specific B-cell memory (Klaus et al., 1980; MacLennan & Gray, 1986
; Tew et al., 1990
). The ability of FDCs to retain native antigens in the form of immune complexes on their surface for considerable periods of time is essential to efficient immune responses within B-lymphoid follicles (Mandel et al., 1980
). The quantity of virus on the FDC surfaces of functional GCs is approximately 1050-fold greater than that of mRNA (Haase, 1999
; Haase et al., 1996
). Accordingly, sequence sets derived from RT-PCR amplification of total RNA effectively correspond to the FDC-borne virus and those derived from spliced mRNA reflect cell-associated mRNA. Hence, it may be concluded that the major fraction of RNA amplified from collagenase-treated cells is indeed coming from FDC-associated virions produced locally.
There exists also a temporal component to the distribution of HIV in GCs. Following multidrug therapy, most FDC-bound HIV decays with a half-life of 1·7 days (Cavert et al., 1997; Hlavacek et al., 1999
). As the majority of this virus is produced in situ, this dynamic process may be accompanied by an equally rapid turnover of CD4 T cells. To this can be added strong immune responses, notably HIV-specific cytotoxic T cells (CTLs), which infiltrate white pulps and GCs (Blancou et al., 2001
; Devergne et al., 1991
; Hosmalin et al., 2001
; Racz et al., 1990
). As the majority of infected cells are depleted before they can produce viruses (Haase, 1999
; Pelletier et al., 1995
), it may well be that, within a GC, viral DNA is turning over faster than viruses on FDC surfaces. In fact, FDC-trapped HIV particles can remain infectious in vivo for more than 9 months (Smith et al., 2001
).
In this respect, inspection of the sequences is revealing. Sequence identity was not always found between the DNA and genomic RNA (e.g. B1, Fig. 1; C19, Fig. 2
). Why should this be, given that transcription is less error-prone than reverse transcription (Mansky & Temin, 1994
, 1995
)? The probability of RNA sequences differing from those of the parent DNA provirus is next to nil. A possible explanation is that viral DNA and genomic RNA are indeed turning over at different rates, such that the provirus that gave rise to FDC-bound HIV had been cleared by the time of sampling. Equally, viral DNA detected could reflect recently immigrated T cells harbouring HIV DNA that are not yet producing virions. The distribution of a fraction of RNA- and DNA-derived sequences in white pulp C19 might well reflect the stark dynamics of infiltration and CTL destruction of infected cells. However, in general, there was correspondence between the dominant RNA- and DNA-derived sequences for all of the white pulps studied.
The findings highlight the very dynamic, yet compartmentalized, nature of HIV replication within lymphoid structures. It follows that models, essential to illuminating such a dynamic process, should take this into account (Grossman et al., 1999, 2002
).
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ACKNOWLEDGEMENTS |
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Received 23 April 2005;
accepted 18 September 2005.
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