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Journal of Virology, May 2002, p. 4379-4389, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4379-4389.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The ITAM in Nef Influences Acute Pathogenesis of AIDS-Inducing Simian Immunodeficiency Viruses SIVsm and SIVagm without Altering Kinetics or Extent of Viremia

Houman Dehghani, Charles R. Brown, Ronald Plishka, Alicia Buckler-White, and Vanessa M. Hirsch*

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852

Received 12 September 2001/ Accepted 29 January 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of the immunoreceptor tyrosine-based activation motif (ITAM) that is unique to the Nef protein of the acutely pathogenic simian immunodeficiency virus SIVsmPBj was studied in the context of two AIDS-inducing simian immunodeficiency virus molecular clones. NefY+ variants of SIVagm9063-2 and SIVsmE543-3 replicated in and induced proliferation of unstimulated pig-tailed macaque PBMC. The pathogenesis of the NefY+ and NefY- clones of SIVagm9063-2, SIVsmE543-3, and PBj6.6 were evaluated by intravenous inoculation of pig-tailed macaques (Macaca nemestrina). Introduction of the ITAM did not increase plasma viral RNA levels nor alter the kinetics of viremia compared with the NefY- versions of each clone. Clinical symptoms were not observed in animals inoculated with the NefY- variants. In contrast, characteristic PBj symptoms were observed in animals inoculated with any of the three NefY+ clones. Blunting and fusion of intestinal villi and multifocal infiltration of mononuclear cells were observed in the gastrointestinal tracts of macaques inoculated with the NefY+ versions. Lesions were associated with active viral replication, as demonstrated by simian immunodeficiency virus-specific in situ hybridization. However, only the macaque inoculated with wild-type NefY+ SIVsmPBj developed fatal disease; lesions were more widespread and severe in this animal. A switch to macrophages as a viral reservoir and the presence of interleukin-6 in plasma was unique to the macaque infected with PBj6.6. Overall, these data suggest that the ITAM in SIV Nef alters the pathogenesis of simian immunodeficiency virus regardless of the viral background. The change in pathogenesis occurs without enhancement of viral replication. However, NefY+ variants of SIVagm and SIVsm did not fully recapitulate the virulence of SIVsmPBj, implicating additional viral factors in this unique virus pathogenesis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Simian immunodeficiency virus (SIV)-induced disease is similar to human AIDS, with the development of high virus loads, progressive depletion of CD4+ T cells, opportunistic infections, and death of infected animals within a few months to years (4). In contrast to the majority of SIV isolates, a virus isolated from a pig-tailed macaque (Macaca nemestrina) infected with the AIDS-inducing SIVsmm9 strain had evolved a variant pathogenesis (5, 10-13). This virus, designated SIVsmPBj14 (for the macaque of origin [PBj] and the month post-SIV inoculation) had evolved the novel ability to replicate in and induce proliferation of unstimulated macaque peripheral blood mononuclear cells (PBMC) in vitro (10). Inoculation of pig-tailed macaques with SIVsmPBj14 induced an acute and lethal illness within 14 days of inoculation, characterized by profuse diarrhea, dehydration, severe lymphopenia, and an extensive cutaneous rash. Pathological features included major gastrointestinal cytopathology with villus blunting (11), massive mononuclear cell infiltration within the gastrointestinal tract, high levels of virus replication in the gut-associated lymphoid tissue (GALT), and immune system hyperactivation (11, 13). Elevated levels of cytokines, such as tumor necrosis factor alpha (TNF-{alpha}) (13, 30) and interleukin-6 (IL-6) (1), produced within the sites of the lesions (4) suggest that the pathogenesis of this novel disease syndrome is cytokine mediated. Evidence of increased apoptosis within gastrointestinal lesions and lymphoid tissues (15) also suggests that apoptotic mechanisms may contribute to the pathogenesis. Interestingly, in light of the gastrointestinal pathology of SIVsmPBj, infection with this virus induces the expression of the mucosal integrin, {alpha}{varepsilon}ß7 in PBMC in vitro and in the tissues of infected macaques. Since this integrin is believed to mediate the mucosal retention of lymphocytes, this effect may be partly responsible for the selective infiltration of lymphocytes in the gastrointestinal sites of infected animals (16).

Studies to delineate the pathogenic determinants of PBj-induced disease have been possible due to the derivation of several molecular clones. One such clone that fully reproduces the pathogenesis of the parental virus is SIVsmPBj6.6 (27). While multiple genes may contribute to the overall virulence of SIVsmPBj, the principle pathogenic determinant appears to be due to a R17Y mutation within Nef. This substitution introduces an immunoreceptor tyrosine-based activation motif (ITAM) into the amino terminus of the SIVsmPBj Nef protein. Introduction of this motif into the Nef protein of the AIDS-inducing SIVmac239 molecular clone confers the ability of the resulting virus to replicate in unstimulated PBMC and to induce acute disease in macaques (8). Conversely, substitution of Y17 in acutely lethal SIVsmPBj clones abrogates the acute pathogenesis (7, 30). This motif is reminiscent of ITAMs in the cytoplasmic domains of B- and T-cell antigen receptors (BCR and TCR, respectively) and Fc receptors (2). ITAMs have also been identified in a number of viral proteins, including EBNA-2 (31) and LMP2A of Epstein-Barr virus (9, 25), K1 of Kaposi sarcoma herpesvirus (22, 23), and gp30 of bovine leukemia virus (21). ITAMs in T and B lymphocytes bind SH-2 domains of Src family kinases. Engagement of the BCR or TCR on lymphocytes upon encountering antigen results in the phosphorylation of both tyrosine residues of the ITAM and activation of signal transduction pathways leading to lymphocyte activation (2). The ITAM of SIVmac239YE Nef was shown to be functionally active for activation of signal transduction, including the activation of ZAP-70 (24). The signaling cascade thus activated by PBj Nef is similar to the events that occur upon TCR cross-linking when T cells encounter antigen. In agreement with this hypothesis, Du et al. (8) determined that SIVsmPBj induces costimulatory pathways in lymphocytes with an apparent requirement for the presence of macrophages in cultures, perhaps to provide a costimulatory signal.

Other studies have identified minor determinants of PBj-induced disease such as duplication of the NF-{kappa}B site in the long terminal repeat (LTR) (3, 5, 26, 27), the U3 LTR promoter region (6), and changes within the viral envelope (27, 29). As expected from their role in AIDS pathogenesis, an intact nef gene (28) and vpx gene (20) are essential for virulence. While the role of Nef may be simply due to the role of ITAMs in acute pathogenesis, Vpx also appeared to be required for replication of the virus in macrophages in vitro and perhaps also in vivo (20). Other substitutions in genes other than nef also appear to have a major effect on virulence of SIVsmPBj6.6. For example, a highly related, minimally pathogenic clone, PBj6.9 shares the tyrosine substitution at position 17 in the Nef protein with the virulent PBj6.6 clone. PBj6.9 differs from PBj6.6 by only one residue in Vpx (C89R), three residues within envelope (D119G, R871G, and G872R) and a single residue in Nef (F252L). A recent study demonstrated that the D119G substitution in gp120 of env of the minimally pathogenic PBj6.9 was responsible for reducing virion infectivity by affecting envelope incorporation into the virus (17). Although introduction of the 6.9 substitution into PBj6.6 (D-119) abrogated pathogenicity, the reciprocal change did not confer full virulence to the parental PBj6.9. This implicates one or all of the other four substitutions relative to PBj6.9 in the virulence of SIVsmPBj6.6. Overall this study suggests that SIVsmPBj is a highly replicative virus, adapted for highly efficeint replication in macaques. Any alterations that alter replicative capacity appear to affect virulence.

In the present study, we confirmed and extended the studies on the role of ITAM in Nef in acute SIV pathogenesis. The ITAM was introduced into Nef of two well-characterized AIDS-inducing SIV molecular clones, SIVsmE543-3 (18) and SIVagm9063-2 (19), by substitution of an arginine at position 17 with tyrosine. In addition, tyrosine 17 of Nef of the SIVsmPBj6.6 clone was substituted with an arginine to inactivate the ITAM. Each of these viruses was studied for its ability to induce proliferation of unstimulated macaque PBMC and replication in macaque monocyte-derived macrophages (MDM), activated PBMC, and unstimulated PBMC. Finally, the pathogenesis of NefY+ and NefY- variants of SIVsmPBj6.6, SIVsmE543-3, and SIVagm9063-2 were evaluated after intravenous inoculation of pig-tailed macaques.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of NefY mutants. The Altered Sites II In Vitro Mutagenesis System (Promega, Madison, Wis.) was used to generate the NefY+ variants of each of the SIV molecular clones. For the mutagenesis of SIVagm9063, the mutagenesis vector pAlter Ex-1 was modified by the addition of an SstI linker into the PstI- and SphI-digested plasmid. The EcoRI-SstI fragment of the full-length SIVagm9063 molecular clone was inserted into the modified pAlter Ex-1 vector. For SIVsmE543-3 mutagenesis, the pAlter Ex-1 vector was modified by the addition of an SfuI linker in the NotI- and NdeI-digested plasmid. AatII-SfuI fragment of the full-length SIVsmE543-3 molecular clone was inserted into the modified pAlter Ex-1 vector. For the SIVPBj6.6 mutagenesis, the pAlter Ex-1 was modified by the addition of a ClaI-SpeI-XhoI linker into the NotI- and NdeI-digested plasmid. The ClaI-XhoI fragment containing the nef gene from PBj6.6 was inserted into the modified pAlter Ex-1 vector. Mutagenesis primers were used to introduce the appropriate substitutions, and the procedure was carried out according to the manufacturer's protocol. The primer for SIVagm9063 mutagenesis was HD1 (5'-GCAAAGCGTGATACAGCGAGACG-3', positions 7942 to 7920), for SIVsmE543-3 the primer was MH1 (5'-GGTGGAAATTTGTACGAGAGACTCTTG CG-3', positions 9120 to 9158), and for SIVPBj6.6 the primer MH2 (5'-GGTGGAAATTTGCGCGA GAGACTCTTGC-3', positions 9097 to 9124) was used.

Each mutated molecular clone was reconstructed by replacement of the appropriate restriction fragment in the original full-length clones with the mutated fragments. Viruses were produced by transfection of 293T cells by the calcium phosphate method (CellPhect Kit; Pharmacia, Piscataway, N.J.). Briefly, cells were plated at a density of 5 x 106 cells 24 h prior to transfection and transfected with 10 µg of plasmid DNA. Transfected cells were maintained in Eagle Minimum Essential Medium (Biofluids, Beford, Mass.) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and HEPES (10 mM). At 72 h posttransfection, culture supernatants were collected and spun at 1,200 rpm for 10 min, filtered with a 0.45-µm (pore-size) filter unit (Millipore, Bedford, Mass.), divided into aliquots, and stored at -175°C. Titers of virus stocks were determined on CEMx174 (SIVsmE543-3 and SIVPBj6.6) or CEMss (SIVagm9063) cells as previously described (18, 19).

Proliferation assay. Pig-tailed macaque PBMC were separated by using lymphocyte separation medium (LSM; ICN Biomedicals, Inc., Aurora, Ohio) according to the manufacturer's instructions. PBMC were suspended in RPMI 1640, supplemented with 10% human AB serum, penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and HEPES (10 mM) at a density of 106 cells/ml. Cells were divided into aliquots and placed into a 96-well tissue culture plate at 100 µl per well and then infected with an equivalent 50% tissue culture infective dose(s) (TCID50) titer of each virus stock or exposed to phytohemagglutinin (PHA; 5 µg/ml) as a positive control for proliferation. After a 5-day incubation period at 37°C, 1 µCi of [6-3H]thymidine (Amersham, Piscataway, N.J.) was added to each well, and the cultures were incubated for an additional 18 h. Cells were harvested, the incorporation of [3H]thymidine was assayed, and the stimulation index (SI) was calculated based upon the mean of the triplicate measurements.

Cell culture and in vitro replication kinetics. Pig-tailed macaque PBMC were separated as described and suspended in RPMI 1640 at 106 cells per ml. Cells were infected with an equivalent TCID50 of each virus for 1 h at 37°C and then washed twice with Hanks balanced salt solution (HBSS). Cells were suspended in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and HEPES (10 mM) and transferred to 12-well tissue culture plates. Supernatant samples were taken at 3-day intervals for 21 days for quantitation of virus replication by using a p27 antigen enzyme-linked immunosorbent assay (ELISA) (Zeptometrix Corp., Buffalo, N.Y.). To measure replication kinetics in activated PBMC, the cells were cultured in RPMI 1640 with 5 µg of PHA/ml supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), HEPES (2 mM), and 10% interleukin-2 (IL-2) for 3 days at 37°C. Prior to infection, the cells were washed twice with HBSS and infected as described above. Cells were maintained in complete RPMI 1640 with 10% IL-2. To derive MDM, PBMC were isolated from the blood of pig-tailed macaques by using Lymphocyte Separation Medium and then cultured for 7 days undisturbed in a T75 flask in RPMI 1640 medium supplemented with 20% FBS and 0.2 ng of granulocyte-macrophage colony-stimulating factor (Sigma, St. Louis, Mo.)/ml. Nonadherent cells were then removed by washing with HBSS and MDM were scraped manually from the plate, and the cells were divided into aliquots in 48-well plates at a density of 106 cells per well by using the media used for original culturing. The cells were allowed to recover overnight and infected with various viruses. For the depletion of monocytes from PBMC cultures, a custom-designed anti-human CD14 tetramer cocktail (Stemcell Technologies, Inc., Vancouver, British Columbia, Canada) was used in the StemSep negative cell selection system (Stemcell Technologies) according to the manufacturer's instructions.

Animal study. Monkeys were maintained in accordance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals. Four groups of three pig-tailed macaques (Macaca nemestrina) each were inoculated intravenously with 2 x 104 TCID50 of SIVagm9063, SIVagm9063Y+, SIVsmE543-3, or SIVsmE543-3Y+. One group of two macaques and another group of one macaque were inoculated with 2 x 104 TCID50 of SIVsmPBj6.6 and SIVsmPBj6.6Y-, respectively. Sequential EDTA-treated blood samples were obtained for lymphocyte subset analysis by flow cytometry (Fast Systems, Rockville, Md.) and quantitation of the viral RNA load in plasma by real-time reverse transcription-PCR (17). The viral load in plasma in macaques was measured with either SIVmac/SIVsm-specific primer or probe (17) for SIVsm-inoculated macaques or SIVagm-specific primer or probe for SIVagm-inoculated macaques (14). IL-6 levels in plasma were assayed by ELISA (Biosource International, Camarillo, Calif.) on samples collected prior to and 8 days after inoculation. Virus isolation was attempted on PBMC samples collected at 1 and 2 weeks postinoculation. One animal from five of the groups was euthanized 8 days postinfection by exsanguination under deep ketamine anesthesia. The animal infected with SIVsmPBj6.6 was euthanized 7 days postinfection due to the severity of disease symptoms. All animals were perfused with heparinized saline, followed by 10% formalin perfusion.

In situ hybridization. Formalin-fixed, paraffin-embedded tissues were stained for SIV viral RNA as previously described (20). SIVsm-specific probes were utilized for SIVsmPBj- and SIVsmE543-infected macaque tissues, and SIVagm-specific probes (19) were used for tissues from animals infected with SIVagm9063. Briefly, the sections were deparaffinized and rehydated with water, pretreated with 0.2 N HCl and proteinase K, prehybridized, and then hybridized overnight at 51°C with either the antisense or the sense riboprobe. The riboprobe consisted of a mixture of probes encompassing 90% of the SIV genome, conjugated with digoxigenin-UTP (Loftstrand Labs, Ltd., Gaithersburg, Md.) at a final concentration of 1.75 ng/µl. The hybridized sections were washed in standard posthybridization buffers and RNase solutions (RNase A [Sigma] and RNase T1 [Roche Molecular Biochemicals, Indianapolis, Ind.]). The sections were blocked in 3% normal sheep and horse serum in 0.1 M Tris (pH 7.4) and then incubated with a 1:500 dilution of sheep anti-digoxigenin-alkaline phosphatase (Roche) for 1 h. Sections were then rinsed in Tris buffer and reacted with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) (Vector Laboratories, Ltd., Burlingame, Calif.) for 10 h and visualized with a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, N.Y.).

Immunohistochemistry and confocal microscopy. Formalin-fixed, paraffin-embedded tissue sections were stained with a rabbit anti-human CD3 antibody, A0452; a monoclonal antibody specific for macrophages, HAM56; and M0632. All antibodies were supplied by Dako (Carpinteria, Calif.). Sections were rehydrated and processed for 6 to 8 min in a Presto pressure cooker (National Presto Industries, Eau Claire, Wis.) in 1 mM EDTA (pH 8.0) to unmask the antigens. The samples were sequentially treated with phosphate-buffered saline, 3% aqueous hydrogen peroxide, serum block (3% normal goat serum, 1% nonfat milk, 0.5% bovine serum albumin), and the specific monoclonal antibody for 1 h. Samples were then rinsed in distilled water and counterstained with hematoxylin. For combined in situ hybridization (ISH) and immunohistochemistry, tissues were hybridized with a digoxigenin riboprobe as outlined above. The hybridized probe was detected with a monoclonal sheep anti-digoxigenin-peroxidase conjugate, followed by fluorescein isothiocyanate-tyramide, NEL 754 (Perkin-Elmer). These samples were stained with either an anti-CD3 (Dako) or anti-macrophage HAM56 (Dako) antibodies and then with either goat anti-rabbit immunoglobulin G-Alexa 633 (A21071) or goat anti-mouse IgM-Alexa 633 (A-21046; Molecular Probes, Eugene, Oreg.), respectively. These double-labeled samples were observed and photographed with a Leica TCS-SP1 confocal microscope.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of Nef mutants of SIVagm9063, SIVsmE543-3, and SIVsmPBj6.6. Previous studies have demonstrated that the unique pathogenesis of SIVsmPBj appears to be associated with the presence of an ITAM within the Nef protein. This ITAM motif [YXXL(X)7YXXL] was created spontaneously in the Nef of SIVsmPBj by a substitution of tyrosine at position 17 for a conserved arginine residue found in other SIV clones. The ITAM motif in Nef of the acutely lethal SIVsmPBj is required for the acute disease phenotype. Thus, introduction of Y-17 into the Nef gene of the AIDS-inducing clone, SIVmac239, alters its pathogenesis in macaques to an acute, diarrheal syndrome similar to that observed following inoculation of SIVsmPBj (8). In contrast mutation of tyrosine-17 of Nef of SIVsmPBj to arginine abrogates its acute pathogenesis (30). To study the role of this motif in the pathogenesis of SIV, the amino acid at position 17 of Nef of two well-characterized, AIDS-inducing molecular clones was mutated to introduce a tyrosine, generating the viruses SIVagm9063Y+ and SIVsmE543-3Y+ (Fig. 1). Thus, an arginine and tryptophan were replaced with a tyrosine by site-directed mutagenesis of SIVsmE543-3 and SIVagm9063-2, respectively. This single substitution introduced an ITAM, the YXXL(X)7YXXL ITAM, into the amino terminus of the Nef protein of these viruses, a result similar to that seen in the SIVsmPBj6.6 molecular clone. The corresponding change of A840S in the Env protein of SIVagm9063 was made without introducing a tyrosine at position 17 of Nef. This virus showed no alteration in its biologic activity compared with the wild-type SIVagm9063 (data not shown). The tyrosine at position 17 of the Nef protein of SIVsmPBj6.6 was mutated to an arginine, yielding the SIVsmPBj6.6Y- molecular clone, which was used as a negative control.



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  		FIG. 1. Schematic diagram of the genome of SIV highlighting the amino terminal region of the nef gene. An alignment of the amino terminus of the Nef protein of SIVsmPBj, SIVsmE543-3, and SIVagm9063-2 is shown below, with the position of the ITAM motif (YXXL X-7 YXXL) in the Nef protein of SIVsmPBj6.6 indicated above the alignment. The alignments of the NefY+ and NefY- variants of SIVsmPBj6.6, SIV smE543-3, and SIVagm9063-2 constructed in this study are shown below.

 
Induction of PBMC proliferation in vitro. The proliferation profiles of the three molecular clones and their respective Nef mutants were compared (Fig. 2A). As expected, SIVsmPBj6.6 induced proliferation of unstimulated PBMC to levels comparable to PHA-induced proliferation. The replacement of tyrosine-17 of SIVsm PBj6.6 with arginine completely abolished its ability to induce activation and proliferation of unstimulated PBMC. The SIVagm9063 molecular clone was unable to induce activation and proliferation of unstimulated PBMC as the SI profile remained the same as for mock-infected PBMC. However, the SIVagm9063Y+ molecular clone induced proliferation of unstimulated PBMC since its SI profile was up to 10-fold higher than the mock-infected PBMC. SIVsmE543-3 also produced an SI profile like that of SIVagm9063. The SIVsmE543-3Y+ was able to induce the activation and proliferation of unstimulated PBMC. The extent of proliferation, observed in a particular experiment, was dependent on the PBMC donor; thus, in experiments carried out with four different macaque donors the SI profile of SIVagm9063Y+ ranged from 5 to 10 (data not shown). The ability of SIVagm9063Y+ to induce the proliferation of unstimulated PBMC from African green monkey (AGM) donors was also evaluated. Although AGM unstimulated PBMC proliferated in response to PHA, no proliferation was observed with either the SIVagmY+ or SIVagmY- cloned viruses (data not shown).



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  		FIG. 2. In vitro properties of Y+ and Y- virus pairs in primary cells. (A) Relative ability of NefY+ and NefY- variants of SIVsmPBj6.6, SIVsmE543-3, and SIVagm9063-2 to induce proliferation of unstimulated pig-tailed macaque PBMC. The SI values are shown graphically, with the standard deviations indicated. (B to D) Replication of SIV variants in unstimulated PBMC (B), PHA-activated macaque PBMC (C), and MDM (D). The NefY- variants are indicated by open symbols, and the NefY+ variants are indicated by filled symbols. Replication was evaluated by SIV p27 antigen in culture supernatant collected sequentially after infection.

 
Virus replication in PBMC and MDM. Previous studies of the parental viruses SIVsmPBj6.6, SIVsmE543-2, and SIVagm9063-2 have demonstrated that each of these viruses replicates in MDM (18, 19) and activated PBMC. However, only SIVsmPBj6.6 replicates in unstimulated macaque PBMC (17). The replication potential of each SIV molecular clone and its respective Nef mutant was analyzed in activated and unstimulated pig-tailed macaque PBMC and macaque MDM. The replication of all six viruses was similar in PHA-activated macaque PBMC cultures as shown in Fig. 2B. In contrast, differential infection was observed after infection of unstimulated PBMC cultures. All of the NefY+ versions of the three virus strains replicated efficiently in unstimulated PBMC, whereas the NefY- versions replicated inefficiently (Fig. 2C). Thus, SIVsmPBj6.6Y- lost the ability to replicate in unstimulated PBMC, although it replicated to the same levels as wild-type SIVsmPBj in PHA-activated PBMC. SIVagm9063 also replicated in PHA-activated PBMC, although it did not replicate to appreciable levels in unstimulated PBMC. The SIVagm9063Y+ virus was able to replicate to levels similar to wild-type SIVsmPBj6.6 in unstimulated PBMC. Similar results were observed with SIVsmE543-3 and SIVsmE543-3Y+ and with SIVmac239 and its NefYE derivative (a gift of R. Desrosiers, data not shown). All six viruses replicated efficiently in macaque MDM (Fig. 2D). However, the NefY+ versions of SIVsmE543 and SIVsmPBj6.6 replicated more persistently and maintained higher levels than the NefY- versions. Replication of the two SIVagm clones was similar.

The ITAM motif of SIVsmPBj is required for acute pathogenicity. Fifteen pig-tailed macaques were inoculated intravenously with 2 x 104 TCID50 of virus. Groups of three macaques each were inoculated with the NefY+ and NefY- versions of SIVagm9063 and SIVsmE543-3. In addition, two macaques were inoculated with SIVsmPBjY- and one was inoculated with SIVsmPBjY+. All animals were monitored for recovery of infectious virus from PBMC cultures, plasma viremia, and lymphocyte subsets. One animal from each group was euthanized on day 8, and tissues were subjected to pathological and virologic analyses. All animals became persistently infected, as determined by the recovery of virus from their PBMC cocultivated with CEMx174 (SIVsm) or CEMss cells (SIVagm).

As expected, the animal inoculated with wild-type PBj6.6 virus (PT 9620) developed diarrhea, generalized skin rash, lethargy, and appetite loss (Table 1). The clinical condition of this animal necessitated euthanasia at 7 days postinoculation. In contrast, no clinical signs were observed in either of the macaques inoculated with the NefY- version of SIVsmPBj (PT 9522 and PT 9527). As shown in Fig. 3A, rapidly increasing levels of viral RNA levels were measured in the plasma of all three macaques, with peak levels of 107 to 108/ml achieved by days 8 to 10 after inoculation. Primary plasma viremia in the macaque inoculated with SIVsmPBj6.6 wild-type virus was indistinguishable from that of the two macaques inoculated with the NefY- version of this clone and from viremia in previous studies of SIVsmPBj6.6 (17). Plasma viremia declined by 1,000-fold by day 21 postinfection of the one SIVsmPBjY--infected animal (PT 9527) allowed to progress past day 8 of infection. Lymphopenia affecting all subsets was observed in all three animals. For macaques inoculated with SIVsmPBjY- virus, lymphocyte numbers declined to ca. 1,000 to 2,000 cells per µl; this was followed in PT 9527 by a rebound of lymphocyte values up to preinoculation levels. Profound lymphopenia was observed only in the macaque (PT 9620) inoculated with wild-type SIVsmPBj. The degree of lymphopenia was compared by using three historical controls that were inoculated previously with SIVsmPBj6.6. As shown in Table 2, the mean lymphocyte numbers at its lowest value were significantly less than in macaques inoculated with the NefY- variant of PBj6.6 (266 compared to 1,204; P = 0.005, Student's t test).


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  		TABLE 1. Summary of clinical findings in SIV-infected macaques

 


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  		FIG. 3. Plasma viremia (top) and absolute blood lymphocyte counts (bottom) in macaques inoculated with SIVsmPBj wild type (open symbols) and SIVsmPBjY+ (filled symbols) (A), SIVsmE543-3 NefY- (B), or NefY+ (C) variants are shown graphically over the first 28 days after intravenous inoculation.

 

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  		TABLE 2. Lymphopenia in macaques inoculated with SIV NefY+ and NefY- virus pairs

 
The ITAM alters the pathogenicity of SIVsmE543-3 and SIVagm9063. A similar dichotomy of clinical signs was observed in macaques inoculated with the SIVsmE543-3 NefY+ and NefY- variants. Characteristic PBj-like signs (rash, diarrhea, and lymphadenopathy) were observed in all three animals inoculated with the NefY+ variant, although the severity of these signs were less than that observed in the macaque inoculated with SIVsmPBj wild type (see Table 1). Considerable animal-to-animal variation in clinical signs was also observed, with only one animal exhibiting the most classic sign of diarrhea (PT 9533). Viral loads in plasma were somewhat lower (105 to 106 copies/ml) and peaked later (14 to 21 days) than those observed in animals inoculated with the PBj clones (Fig. 3B and C). However, the kinetics of plasma viremia in the animals inoculated with the wild-type SIVsmE543 NefY- were remarkably similar to those observed in animals inoculated with the NefY+ variant. The one exception was PT 9525, inoculated with SIVsmE543Y+, in which primary viremia was extremely low. All of the animals exhibited lymphopenia regardless of which variant of SIVsmE543 they received (Table 2). Lymphopenia was generally more severe in macaques inoculated with the NefY+ variant (a mean of 844/µl) compared to those inoculated with the NefY- variant (1,392/µl), although the difference was not statistically significant (P = 0.07 by Student's t test).

As observed with the SIVsm virus pairs, no clinical signs were observed in macaques inoculated with the SIVagm9063 NefY- variant. In contrast, two of the three macaques inoculated with the NefY+ variant exhibited a combination of typical PBj-like signs (rash, anorexia, and diarrhea; Table 1). Plasma viremia peaked at 107 to 108 copies/ml by day 8 postinoculation in both groups of animals. As observed in the SIVsm virus pairs, the kinetics of viremia were essentially identical in the two groups of animals. All macaques also exhibited lymphopenia by day 4 to 8 postinoculation (Fig. 4). The mean lymphopenia in the macaques inoculated with the NefY+ variant was more severe than that observed in those inoculated with the NefY- variant (862 and 1,519 cells per µl, respectively; Table 2). This difference was statistically significant (P = 0.02, Student's t test). However, lymphopenia observed in macaques inoculated with SIVsmPBj6.6 was significantly more severe than in any of the other groups.



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  		FIG. 4. Plasma viremia (top) and absolute blood lymphocyte counts (bottom) in macaques inoculated with SIVagm9063-2 NefY- (A) or NefY+ (B) variants are shown graphically over the first 28 days after intravenous inoculation.

 
The Nef ITAM motif alters gastrointestinal pathology. The histopathologic findings in the six macaques euthanized at day 8 of infection were evaluated and compared between virus pairs. Pathological changes were restricted to the gastrointestinal tract. As expected, SIVsmPBj induced characteristic gastrointestinal changes, including blunting and fusion of intestinal villi, and massive infiltration of mononuclear cells into the lamina propria and GALT (Fig. 5A). In contrast, the gastrointestinal tissues from the macaques inoculated with the NefY- variant of SIVsmPBj were within normal limits, with normal villus architecture and GALT structure (Fig. 5B). The gastrointestinal histopathology of macaques inoculated with the parental SIVsmE543-3 and SIVagm9063-2 were also within normal limits (Fig. 5C and E). However, intestinal pathology was observed in both macaques inoculated with the NefY+ variants of both SIVsmE543 and SIVagm9063. Characteristic blunting and fusion of intestinal villi and infiltration of mononuclear cells into the lamina propria were observed in both animals. In addition, massive hyperplasia and expansion of GALT was observed in the macaques inoculated with the NefY+ variant of SIVagm9063 (Fig. 5D and F). The lesions were more focal and less severe than those observed in the macaque inoculated with PBj.



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  		FIG. 5. Histopathologic changes in hematoxylin- and eosin-stained sections of the ileum from macaques inoculated with NefY+ (A, C, and E) and NefY- (B, D, and F) variants of SIVsmPBj6.6, SIVagm9063-2, and SIVsmE543-3, respectively. Normal villus architecture was observed throughout the gastrointestinal tract in macaques inoculated with NefY- variants. Various degrees of mononuclear cell infiltration, villus blunting and fusion, and GALT expansion were observed in macaques infected with the NefY+ versions of all three clones, although the most severe lesions were observed in macaques inoculated with wild-type SIVsmPBj. Magnification, x10.

 
Lymphoid and intestinal tissues were also evaluated by SIV-specific ISH. The number of SIV-expressing cells was highest in macaques inoculated with the PBj6.6 viruses, intermediate in SIVagm-inoculated macaques, and lowest in frequency in SIVsmE543-3-inoculated macaques. As expected from the viral loads in plasma, the numbers of SIV-positive cells were similar in animals infected with each virus and its respective NefY variant. For each of the virus pairs, SIV-positive cells were observed in peripheral and mesenteric lymph nodes, spleen, and thymus in similar numbers and distribution (data not shown). For instance the numbers of virus infected cells in the tissues of the animals infected with SIVPBj6.6NefY- were similar to those of the SIVPBj6.6-infected animals, as were their virus loads in plasma. The same was true for both SIVsmE543-3 and SIVagm9063. However, the distribution and number of virus-expressing cells differed significantly in the gastrointestinal tract (Fig. 6A and B). SIV-positive cells were observed in the GALT and in the tips of villi of intestinal tissues of the macaque inoculated with the NefY- variants (SIVagm shown in Fig. 6C and D). In contrast, large numbers of virus-expressing cells were diffusely distributed throughout the lamina propria and GALT of the intestinal tract of the macaque inoculated with NefY+ variants. The highest density of SIV-positive cells were observed in sites of characteristic intestinal pathology, such as villus blunting, fusion, and erosion.



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  		FIG. 6. ISH for SIV expression in macaques infected with NefY+ and NefY- versions of SIVsmPBj6.6 (A and B) and SIVagm9063 (C and D). The top panels show representative ISH of the ileocecal junction of the intestine of PBjY+ (A) and PBjY- (B). The bottom panels show the ileocecal junction of the intestine of animals infected with SIVagm9063Y+ (C) and SIVagm9063Y- (D). All sections were stained as described in Materials and Methods and were counterstained with eosin. Magnification, x20.

 
Similarities and differences compared to wild-type SIVsmPBj infection. Tissues of animals inoculated with each of the clones were evaluated for some of the hallmarks of PBj-induced disease, including in vivo evidence of infection of macrophages, activation markers, and cytokine levels. Sections of mesenteric lymph node from macaques inoculated with each version of the three viruses were examined for evidence of in vivo proliferation as assessed by the nuclear expression of Ki-67 antigen. The number of Ki-67+ cells in the paracortical regions of lymph nodes from Y+-inoculated macaques was significantly higher than that observed in their respective parental control (2.6-fold higher for the SIVagm and 4.3-fold higher for SIVsmE543). However, the numbers of Ki-67+ lymphocytes in the nodes from the SIVsmE543 (175/high-power field [hpf]) and SIVagm (193/hpf) NefY+-inoculated macaques were considerably lower than in the PBj6.6 Y+-infected macaque (290/hpf).

Since we observed a difference in the ability of the NefY+ variants to replicate in MDM in vitro, we also were interested in the types of cells infected in vivo. SIV-positive cells in the macaque infected with the PBj NefY- virus were compact in morphology, which is typical of lymphocytes. In contrast, a number of multinucleated cells and cells with extensive cytoplasm, which is reminiscent of macrophages, were observed in the wild-type PBj-infected animal. Confocal microscopy of the mesenteric node and intestine confirmed that SIV-positive cells in the macaque inoculated with PBj6.6NefY+ consisted of a mixture of macrophages and CD3+ T cells (Fig. 7). In contrast, SIV-positive cells in tissues of the macaque inoculated with PBj6.6Y- were exclusively CD3+ T cells (data not shown). Tissues from the macaques inoculated with SIVagmY+ and SIVagmY- variants were also evaluated by confocal microscopy. The majority of SIV-positive cells in these animals appeared to be CD3+ T cells with few if any SIV-positive macrophages observed (data not shown), regardless of whether the animal had been infected with the NefY+ or NefY- variant.



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  		FIG. 7. Confocal micropscopy of mesenteric lymph node sections from macaques infected with PBjY+. The top panels show the same fields double stained with SIV riboprobe (green) and CD3 (red) and the merged view. The bottom panels show another field stained with SIV riboprobe and Ham56. SIV-expressing cells that coexpressed either CD3 (T cells) or Ham56 (macrophages) were identified.

 
Previous studies of SIVsmPBj demonstrated elevations in plasma cytokines, particularly IL-6. Thus, plasma samples collected preinoculation and 7 days postinoculation were analyzed for IL-6 by ELISA. Massive elevation (100-fold over preinoculation values) in IL-6 levels was observed in the plasma of the animal inoculated with wild-type SIVsmPBj (580 pg/ml). No change in IL-6 levels were observed in the macaques inoculated with the NefY- variant of SIVsmPBj or in any of the other study animals.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study extends findings on the introduction of the Nef ITAM into SIVmac239 (8) to two additional AIDS-inducing SIV clones and confirms that this motif is responsible for the unique pathogenesis of SIVsmPBj. As expected, the ITAM was essential for the ability of these viruses to induce proliferation of unstimulated PBMC and enable them to replicate in unstimulated macaque PBMC but did not affect their ability to replicate in PHA-activated PBMC. This result differs from the report by Saucier et al. (30) in which growth in unstimulated PBMC was unaffected by removal of the ITAM from Nef of SIVsmPBj but agrees with the report by Du et al. (7). The presence of the Nef ITAM also enhanced their ability to replicate in MDM. The ITAM was required for the acute pathogenesis of SIVsmPBj6.6 and altered the pathogenesis of two AIDS-inducing SIV molecular clones, SIVsmE543-3 and SIVagm9063. The alteration in the pathogenesis SIVsmE543-3 from the same phylogenetic lineage as SIVmac239 was not unexpected. Although these viruses are similar in pathogenicity in vivo, SIVsmE543-3 and SIVagm9063 replicate in both lymphocytes and macrophages in vitro, whereas SIVmac239 is strictly lymphocyte-tropic. Surprisingly, introduction of the ITAM into the highly divergent nef gene of SIVagm9063, a virus from a separate lineage from SIVsm/mac also resulted in a syndrome similar to that observed with SIVsmPBj. Whereas the gastrointestinal tissues from macaques inoculated with the NefY- variants had normal villus architecture and GALT structure, macaques inoculated with the NefY+ variants exhibited PBj-like lesions. Interestingly, the introduction of the Nef ITAM did not enhance viral replication in vivo, as assessed by both viral RNA levels in plasma and the numbers of SIV-expressing cells in tissues. Therefore, the altered pathogenesis induced by the ITAM is not the result of simply increasing the number of activated T-lymphocyte target cells for virus replication. More likely, the nefY allele alters the trafficking of infected cells and interacts specifically with T-cell signaling proteins in the sites of gastrointestinal lesions to promote the influx of lymphocytes and macrophages into sites of SIV replication in the gastrointestinal tract.

Despite an obvious shift in the pathogenesis of SIVagm9063Y+ and SIVsmE543-3Y+ from their parental strains, the lesions in macaques inoculated with these viruses were less severe than observed in macaques inoculated with SIVsmPBj6.6. Interestingly, we were able to recreate more closely the PBj-like phenotype in the SIVagm9063 clone, which is phylogenetically more distant from SIVsm, suggesting that the role of Nef in pathogenesis is conserved among divergent primate lentiviruses. The kinetics of replication of the SIVagm clones in macaques more closely paralleled that of SIVsmPBj, with more rapid kinetics than were observed in SIVsmE543-3-inoculated macaques, suggesting that rapid viral kinetics is an essential feature of this disease syndrome. This study clearly demonstrates that the presence of the ITAM motif in Nef of SIVsmE543-3 and SIVagm9063 is sufficient and necessary to induce histopathologic lesions similar to observed with SIVsmPBj6.6. However, as previously reported with SIVmac239 NefYE (7, 8), neither of these NefY+ clones reproduced the full virulence of wild-type SIVsmPBj. This suggests that other viral factors unique to SIVsmPBj are required to reach the full acute pathogenic potential. The viral genes and pathogenic processes that differ are not clear from this study. Two features of wild-type SIVsmPBj infection appeared to be unique to this virus: in vivo infection of macrophages and elevated levels of IL-6. However, it is not clear whether these are causative or secondary features.

Macrophages comprised the majority of infected cells in tissues analyzed 8 days postinoculation obtained from animals inoculated with SIVsmPBj6.6. This finding was in clear contrast to that for tissues from macaques inoculated with SIVsmPBj6.6Y- or either SIVagm variant,where T cells comprised the majority of infected cells. In vivo macrophage infection was clearly not essential for the development of initial gastrointestinal lesions since lesions were observed in SIVagmY+-infected macaques in the absence of demonstrable infected macrophages. However, in vivo infection of macrophages may be associated with progression to more-erosive fulminant lesions observed only in macaques inoculated with wild-typePBj6.6. It is also possible that the infection of macrophage population observed exclusively in PBj6.6 may be a feature of the end-stage of disease. Euthanasia of the macaques was not timed for the severity of clinical symptoms, and therefore the macaque inoculated with PBj showed more severe symptoms than the SIVagmY+-inoculated macaque at the time of euthanasia. A similar switch into macrophages might have been observed if the SIVagmY+-inoculated macaque had been euthanized a day or two later. Interestingly, SIVmac239YE does not infect macrophages in vitro and produced a similar partially virulent phenotype in vivo (8), a finding similar to that observed in our SIVsm and SIVagm clones. The large numbers of infected macrophages in SIVsmPBj6.6-inoculated animals may result in chemotaxis of T lymphocytes to the intestinal tract and activation of local T-cell populations and provide source of proinflammatory cytokines such as TNF and IL-6. Indeed, the other unique feature observed in the SIVsmPBj6.6-inoculated macaque was elevated levels of IL-6 in plasma. These two features may be linked since IL-6 is a known product of macrophages. Further studies will be required to determine whether these factors contribute to the full pathogenic phenotype of SIVsmPBj and to identify other critical viral genes.


   ACKNOWLEDGMENTS
 
We thank R. Byrum and M. St. Claire of Bioqual, Inc., for assistance with the animal studies; O. Schwartz of RTB, NIAID, for assistance with confocal microscopy; and S. Goldstein, S. Whitted, and R. Goeken for technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Twinbrook II Facility, 12441 Parklawn Dr., Rockville, MD 20852. Phone: (301) 496-2976. Fax: (301) 480-2618. E-mail: vhirsch{at}nih.gov. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, May 2002, p. 4379-4389, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4379-4389.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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