INTRODUCTION

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Genetic variation of the lentiviruses is thought to play a major role in transmission (42), development of immunodeficiency (37, 41) and neurological disease (33), escape from host defenses (39), and resistance to antiviral drugs (28). Differences, for example, in regions of the lentivirus env gene that are correlated with the ability to replicate in cultured macrophages are also correlated with the macrophage tropism of virus strains that successfully transmit infection (22) and with replication in macrophages and microglia in the nervous system (16). In later stages of infection, evolution of genetic diversity in the env gene of human and simian immunodeficiency viruses (HIV and SIV, respectively) is associated with poor growth in cultured macrophages but rapid growth with induction of syncytia (syncytium-inducing strains) in cultured lines of CD4⁺ T lymphocytes and with the development of immunodeficiency (40).

The genetic diversity of lentiviruses circulating in the bloodstream or in tissues or tissue fluids has thus far been characterized by sequencing portions of env amplified from extracted nucleic acids (6, 24) and by heteroduplex mobility analysis (13). Much has been learned in this way about populations of lentiviruses, but there is little understanding as yet of the interactions in vivo between the individual members that comprise quasispecies and their host cells. To address this gap in our understanding of lentivirus pathogenesis, we have devised and describe in this report an in situ hybridization (ISH) approach to investigating the replication of individual genotypes that defines cellular and tissue tropisms and sites of replication at single-cell resolution.

	MATERIALS AND METHODS

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Animals and virus. Rhesus macaques used for these studies were housed at the BIOQUAL animal facility (Rockville, Md.), and all animal experiments were performed in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals. Detailed descriptions of the animals, clinicopathological findings, and ISH findings with representative probes are presented separately (36). Briefly, juvenile rhesus macaques were inoculated with 10 rhesus macaque infectious doses of cell-free SIVdeltaB670 grown in human peripheral blood mononuclear cells (34). This inoculum was an aliquot of the same stock used previously (34) that on repeated passage in peripheral blood mononuclear cells maintains the same major genotypes and predominant clonotypes (clones 3 and 12 [see below] as the parent (3). Prior to sacrifice, animals received ketamine (20 mg/kg) and were subsequently anesthetized with ketamine-acepromazine (10 mg/kg). Animals were then perfused transcardially with phosphate-buffered saline (PBS; approximately 2 liters/kg of body weight), 1% formalin in PBS (400 ml/kg), and 4% formalin in PBS (approximately 1.5 liters/kg). Tissue specimens were removed and fixed overnight in 4% paraformaldehyde-PBS, cryopreserved by sequential immersion in 10 to 20% sucrose in PBS over 48 h, and snap frozen in isopentane cooled to 70°C.

ISH. ISH for detection of SIV RNA with antisense oligonucleotide probes was performed as described previously (36). Briefly, 14-µm sections were cut from cryopreserved tissues, thaw mounted onto 3-aminopropyltriethoxysilane-coated microscope slides, and stored at 70°C until use. Sections were then postfixed in 4% paraformaldehyde-PBS for 20 min, washed in 70% ethanol for 20 min, and dehydrated in graded ethanols. Pretreatments consisted of either of two regimens, both providing equivalent ISH results (data not shown). The first pretreatment consisted of incubation for 20 min each, in 0.2 N HCl at ambient temperature, with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 70°C and 2 mM CaCl₂-20 mM Tris (pH 7.5)-10 µg of proteinase K per ml at 37°C; two 5-min washes in diethyl pyrocarbonate (DEPC)-treated distilled H₂O (dH₂O); and acetylation in 0.25% acetic anhydride-0.1 M triethanolamine (pH 8.0). The second pretreatment protocol is described in the description of combined ISH and immunuhistochemical (IHC) staining. Following dehydration in graded ethanols, 300,000 to 500,000 cpm of ³⁵S-labeled probe in 10 µl of hybridization mix (35) was spread on the section under a coverslip, which was then sealed with rubber cement and hybridized at 37°C for 18 h. Sections were then washed twice through 2× SSC (room temperature, 5 min), once through 0.2× SSC (room temperature, 5 min), once through 0.2× SSC (54°C, 1 h), and extensively (2 days) in hybridization wash medium (0.6 M NaCl, 10 mM Tris [pH 7.4], 1 mM EDTA, 5 mM dithiothreitol, 50% formamide). Sections were dehydrated in graded ethanols containing 0.3 M ammonium acetate, air dried, coated with NTB-2 emulsion (Kodak, Rochester, N.Y.), and exposed at 10°C for 3 to 14 days. Oligonucleotide probes were 3'-end labeled with [³⁵S]dCTP (NEN/DuPont), using terminal deoxynucleotydaltransferase, to specific activities of 5 × 10⁹ to 8 × 10⁹ cpm/µg.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   SIV genotype-specific oligonucleotide probes. (A) Alignment of the V1 regions of SIVdeltaB670 clones 2, 3, 6, and 12 reported by Amedee et al. (3). Dashes represent sequence identity with clone 2, dots represent sequence gaps, and lowercase letters represent nucleotide insertions. The regions to which the genotype-specific probes were complementary are boxed. (B) Sequences and properties of SIV genotype-specific probes. The melting temperature (Tm) was calculated as follows: 16.6 log10 [Na+] + 81.5 + 41.5 [% GC]  675/length  % mismatch  0.65 [% formamide], for hybridization in 0.6 M NaCl and 45% formamide, length of 30 or 32, and 0% mismatch.

Plasmids and transfections. Plasmids containing the 1.9-kb env inserts from clones 2 to 12 (3) were restriction digested with EcoRI. Agarose gel-purified env inserts were then ligated to the eukaryotic expression vector pCMV-5 (4).

Combined ISH and IHC staining for cell type markers. To unambiguously identify the cell types expressing SIVdeltaB670 RNAs, ISH and IHC were performed on the same tissue sections. Tissue sections were heated in a microwave oven in 0.01 M citrate buffer (pH 6.0) for 10 min at 800 W with interruptions at 1- to 2-min intervals to replace lost liquid. Sections were cooled at room temperature for 30 min, rinsed in DEPC-treated dH₂O, acetylated, and dehydrated in graded ethanols. After ISH and stringent hybridization washes, sections were rinsed in 1× PBS twice for 5 min. Nonspecific antibody binding sites on sections were blocked by incubation for 1 h in 1× PBS with 5% nonfat dry milk (PBS-NFDM). Excess blocking solution was wiped away and replaced with antigen-specific primary antibody for 1 h. Sections were washed in 1× PBS twice for 5 min each time, and cells to which the primary antibodies bound were detected by the avidin-biotin complex method (ABC kit; Vector Laboratories) with 3,3'-diaminobenzidine as the substrate. The sections were coated with NTB-2 emulsion (Kodak), exposed, developed, and counterstained lightly with hematoxylin. All reagents for combined ISH and IHC were made with DEPC-treated dH₂O or were treated directly with DEPC. All incubations for IHC were performed at room temperature, and all diluents were PBS-NFDM. Primary antibodies were specific for CD68 (murine monoclonal KP1; Dako) or CD3 (rabbit polyclonal serum; Dako).

RT-PCR and sequence analyses. Nested reverse transcription (RT)-PCR for plasma viral RNA, subcloning, and DNA sequence analyses were performed as described previously (3). Pairwise nucleotide sequence comparisons of V1 clones 2 to 12 and genotype-specific probes were performed with the EGCG extensions (Peter Rice, The Sanger Centre, Cambridge, England) to the Wisconsin package (Genetics Computer Group, Madison, Wis.).

	RESULTS

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To analyze the replication of individual genotypes in vivo, we designed genotype-specific probes that could be hybridized in situ to detect expression of particular genotypes in cells in tissue sections. We developed and applied this method in an experimental model of lentivirus infection that has relevant and important parallels to HIV infection, including immunodeficiency and neurological disease (15). A cohort of juvenile macaques was inoculated with a pathogenic isolate of SIV (deltaB670 [34]). Sequences in the first and most variable region (V1) of the env gene have been determined previously in a separate stock of SIVdeltaB670 from which the stock used here was expanded and in viruses in the blood and tissues of infected animals (3). We focused on V1 initially because it differs to the greatest extent between genotypes and is a major determinant of tropism (2, 30, 32, 37). Although there are multiple V1 genotypes in SIVdeltaB670 isolates, we concentrated on four V1 clones (clones 2, 3, 6, and 12) because they represent 88% of the V1 sequences identified in infected animals and 100% (clone 12) of the V1 sequences transmitted in utero (3).

We aligned the V1 sequences of these four clones to identify the regions of greatest diversity, such as regions with insertions or deletions, among the different genotypes (Fig. 1A), and we designed and synthesized antisense oligonucleotide probes of 30 to 32 nucleotides targeted to these regions. The four probes were comparable in GC content and melting temperature (Fig. 1B), were similar in size, and could be labeled to specific activities equivalent to those of probes that we have used to detect spliced and unspliced SIV RNAs in tissues (35). We documented the specificity of these probes in COS cells transiently transfected with eukaryotic expression vectors containing recombinant inserts derived from the env clones. After ISH and autoradiography, the hybridization signal of hundreds of (black) silver grains over background was limited to cells transfected with the cognate target sequences (Fig. 2). There was no appreciable binding of probes to cells expressing heterologous V1 sequences with up to 93% sequence identity, even though env sequences from clones 2 through 12 were expressed at high levels in the transfected COS cells and could be detected with a riboprobe complementary to the env gene (not shown).

View larger version (98K): [in this window] [in a new window]   FIG. 2.   Specific ISH detection of SIV RNAs with antisense oligonucleotide probes. Plasmids expressing the env fragments from the indicated SIVdeltaB670 clone were electroporated into COS cells and env V1-targeted antisense oligonucleotide probes were hybridized in situ to expressed RNAs. Autoradiographs were exposed for 1 day. Magnification, ×78. The percent sequence homology is for the best-matched 30-nucleotide target in the corresponding V1.

We used the genotype-specific probes in ISHs to identify the cells and anatomic sites of replication of each of the four genotypes in six rhesus macaques inoculated with uncloned SIVdeltaB670. The animals were euthanized 2.5 to 6 months postinoculation, when they developed AIDS, or at 6 months postinoculation in a control, infected asymptomatic group. We detected, by ISH with riboprobes complementary to pol and env, cells with abundant viral RNA in lymphoid tissues, nervous system, lung, and gut, as well as viral RNA in virions on the surfaces of follicular dendritic cells in germinal centers (Table 1). Peripheral and tissue viral loads were uniformly higher in all tissues in the animals that developed AIDS (36).

The four V1 variants replicated to different extents and with distinctive patterns within and between animals, and in general, the most prevalent genotypes in the tissues were also the most prevalent in plasma (Table 1). The discrete microanatomical sites of replication and relative prevalences of the four V1 variants in spleen are illustrated in Fig. 3. Clones 2 and 6 were found more frequently in the macrophage-rich red pulp areas, whereas a much greater proportion of cells infected with clone 3 or 12 variants was found in the T-lymphocyte-rich periarteriolar lymphoid sheaths of the white pulp, although replication was not restricted to white pulp. This was one indication that these variants were dual tropic in vivo, a finding which we document more directly below. V1 clones 3 and 12 replicated in more cells and organ systems, and to higher levels in the animals with AIDS, than clone 2 or 6, and clone 3 was the only variant that replicated in the asymptomatic animals, with the exception of rare cells in the thymus in which we detected clone 12 RNA (Table 1). These findings are consistent with the observation that clones 3 and 12 are the most frequently found variants in infected animals (3) and with the enigmatic association of clone 3 with infections that progress slowly to manifest illness (2a).

View larger version (98K): [in this window] [in a new window]   FIG. 3.   Spatial distribution of cells productively infected with SIVdeltaB670 variants in spleen tissue from animal MO74. Subjacent tissue sections were hybridized with the four genotype-specific probes (Table 1), and the gross locations of the foci of infected cells (e.g., insets) detected with individual probes are indicated by the overlaid colors.

Clonotypic viral gene expression also occurred in spatially restricted and defined patterns in nonlymphoid tissues; for example, we found many cells with high levels of clone 12 RNA in regions of the brain or gut in which there was no evidence of replication of clone 3 (or clones 2 and 6) (Fig. 4). Levels of viral gene expression were comparable between genotypes and comparable to those determined for sequences surrounding the major subgenomic splice donor, 1,200 copies of RNA/cell (35). This we determined by counting silver grains over cells by quantitative image analysis (23), and we found on average that there were approximately 1,600 copies of V1 clonotypic RNA per cell (not shown).

View larger version (95K): [in this window] [in a new window]   FIG. 4.   Replication of V1 clones 3 and 12 at discrete sites in the gut and brain in animal M086. ISH to subjacent tissue sections with the antisense V1.clone.3 or V1.clone.12 probe demonstrated clone 12 replication in the shown regions of the tissue specimen. Autoradiographs were exposed for 7 days. Magnification, ×44.

To address the issue of the relationship of genetic diversity to cellular tropisms in vivo, we combined ISH with genotype-specific probes and IHC staining to identify the types of cells infected by the variants. Infection with clone 12 has previously been associated with encephalitis and AIDS; clone 12 is the predominant viral strain isolated from infected tissues and replicates in vitro in primary rhesus macrophages (3). We determined the cellular tropisms of clone 12 and clone 3 in vivo by staining cells in the macrophage lineage with antibody to CD68 and staining T lymphocytes with antibody to CD3. In the central nervous system, most productively infected cells have been shown to be macrophages and microglia (8, 27), and not unexpectedly, we detected high levels of clone 3 and clone 12 RNA in cells that stained with anti-CD68 antibody (Fig. 5). There were, however, occasional infected CD³⁺ T lymphocytes (not shown). In lymphoid tissues and gut, we found evidence of viral replication of both clones in T lymphocytes (Fig. 5) and in macrophages (not shown). We thus document by double-label ISH with genotype-specific probes that the predominant replicating variants in the SIVdeltaB670 isolate are dual tropic in vivo.

View larger version (89K): [in this window] [in a new window]   FIG. 5.   Dual tropism of V1 clones 3 and 12. In the central nervous system (CNS) or gastrointestinal tract, macrophages (MØ) were identified by IHC staining with anti-CD68 antibodies, and T lymphocytes were detected with anti-CD3 serum. Clonotypic viral RNA was similarly detected with the genotype-specific probes. Autoradiographs were exposed times for 12 to 14 days. Original magnification, ×122 to ×190.

	DISCUSSION

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We have shown for the first time that individual, expressed genotypes comprising a primate lentivirus quasispecies can be tracked at single-cell resolution in tissue sections. SIVs harbored within productively infected cells in tissues, whose V1 sequences were more than 90% homologous, were distinguished in vivo to reveal their cellular and anatomic sites of replication. Previously, DNA sequence analysis of HIV type 1 proviral DNA in individual microdissected splenic white pulps provided evidence of spatial compartmentalization of HIV genotypes (10) and suggested that local foci of similar genotypes arose due to the recruitment and proliferation of antigen-specific T lymphocytes. We now document compartmentalization that extends to the level of individual cells expressing viral RNA and antigen (not shown) in the spleen and other organs that can readily account for founder effects and differences in lentivirus quasispecies between and within organ systems in studies limited in analysis to viral DNA (10, 12, 20, 38).

In addition to spatially defined patterns of replication within an organ, widespread dissemination also varied between the V1 genotypes. Only the two highly virulent clone 3 and 12 variants were widely disseminated, and the cells productively infected with these viruses comprised the vast majority of those expressing viral RNA (data not shown). The predominance of these clonotypes in infection with an inoculum obtained by passage of the original stock (3) attests to the replicative fitness of these two variants. The other members of this quasispecies did not disseminate throughout the animals despite replication in the spleen and the immunocompromised status of the animals and, therefore, presumably permissive environment for viral dissemination. It is possible that local, residual immune responses to specific viral epitopes expressed by certain env genes contributes to the lack of dissemination of genotypes other than clones 3 and 12. Alternatively, there may be depletion of a specific subset of host target cells that these variants require for propagation, or there may be restrictions on the abilities of some variants to use alternate entry cofactors such as the -chemokine receptors (1, 11, 14, 17, 18, 21). In support of this view, the expression of the CCR5 and CXCR4 coreceptors is differentially regulated on naive and memory T lymphocytes (5) and on T lymphocytes simultaneously stimulated through CD3 and CD28 (7).

We did not observe tissue-specific expression of viral genotypes except in the spleen, where there was evidence of replication of all four variants (Table 1 and Fig. 3). This was nonetheless insufficient to alter the predominance of clones 3 and 12 in the plasma or in other tissues (Table 1). It is possible that further PCR analyses of peripheral blood and proviral DNA from tissues will detect replication of other variants such as clones 2 and 6.

Our observation that both of the pathogenic variants, clones 3 and 12, were dual-tropic for macrophages and T lymphocytes suggests that their abilities to use coreceptors on these alternate cell types contributes to their ability to replicate to high levels and disseminate widely. Specific changes in their env sequences might allow the use of not only CCR5, which is used by all SIVs analyzed to date (9, 19, 29), including clone 3 (19), but also other coreceptors, an issue that we are now investigating. In this study as well as others (3), clone 3 is a predominant replicating variant, but it also is the most frequently detected genotype in asymptomatic animals (Table 1; reference 2a). This dual phenotype implies the involvement of either additional viral determinants of outcome outside the V1 region of env or different phenotypes dependent on host factors. We cannot rule out the possibility that changes outside V1, which our probes will not detect, account for the dual tropism of clones 3 and 12 or the dual phenotype of clone 3. It will be necessary to examine a larger number of animals and animals in the acute phase of infection to ascertain whether the dual tropism of these variants is an inherent biological feature or whether it evolves during the course of infection in vivo. With the experimental approach that we describe here, it should be possible to investigate how interactions between cells and viruses with defined differences in specific regions of env and other genes affect viral transmission, dissemination, pathogenesis, and response to treatment.