Infection of rhesus macaques with chimeric simian-HIV (SHIV) has proven invaluable in providing insights into the in vivo functions of various HIV gene products and in evaluating potential HIV therapies and vaccines.1-4 In particular, the SHIV macaque model has demonstrated the importance of HIV-1 envelope (Env) glycoproteins in mediating viral pathogenesis.5-8 In vivo passage-associated mutations in env gp120 are sufficient to confer pathogenic phenotypes to CXCR4 (X4) using SHIV molecular clones, and such mutations have been associated with enhanced viral entry, cell killing, and immune escape of the virus.6,9,10 It is R5 viruses that are mostly associated with mucosal transmission and acute infection,11 however, and studies of the genetic and phenotypic determinants of pathogenesis of such SHIVs have been more limited. Understanding Env-mediated functions in the context of R5 SHIV molecular clones should facilitate the development of prophylactic or therapeutic interventions that protect against sexual transmission and contain early stages of HIV infection.
To this end, rapid serial in vivo passage of an R5 clone virus SHIVSF162 was used to derive SHIVSF162P3, a pathogenic and vaginally transmissible isolate that maintained CCR5 use.12,13 To define and characterize the adaptive mutations responsible for the phenotype of the isolate, we constructed SHIVSF162PC using the dominant env gp120 sequence of SHIVSF162P3.14 P3gp120 has 14 amino acid changes relative to the SF162 gp120 sequence, and although SHIVSF162PC reproduced the mucosal transmissibility of SHIVSF162P3, it did not induce disease. In an effort to generate an infectious pathogenic R5 SHIV molecular clone, we amplified full-length P3gp160 by reverse transcriptase polymerase chain reaction (RT-PCR) and constructed a molecular clone, SHIV P3gp160. In this study, we assessed the mucosal transmissibility and replicative ability of the SHIV P3gp160 molecular clone in rhesus macaques.
Reverse Transcriptase Polymerase Chain Reaction Cloning of P3gp160 Sequence
Viral RNA was prepared from 500 μL of plasma using a commercially available RNA extraction kit (Qiagen, Chatsworth, CA) and was resuspended in 50 μL of double distilled H2O (ddH20). This is the same plasma sample that was used to amplify the P3gp120 coding sequence previously described.14 RNA was reverse-transcribed with Superscript RT (Life Technologies) and a random hexamer primer (Amersham Pharmacia, Piscataway, NJ), and P3gp160 complementary DNA (cDNA) was amplified with a nested PCR assay.15 Briefly, a 50-μL reaction containing 2 to 5 μL of template cDNA, 2.5 U of Expand polymerase (Roche, Mannheim, Germany), Expand buffer 3, and 500 μM each of a deoxynucleoside triphosphate (dNTP) cocktail (Amersham Pharmacia) was prepared. The reaction cocktail contained 200 nM each of primers 989L (sense)15 and P2016 (TTG TGC TTC TAG CCA GGC ACA ATC, antisense). The PCR assay was carried out for 30 cycles at 92°C for 45 seconds, 45°C for 45 seconds, and 68°C for 300 seconds. Two microliters from the first PCR assay was subsequently used in a second 20-μL reaction, which consisted of similar reaction buffers and enzyme but with 200 nM of primers 944S (AGA AAG AGC GGC CGC CAG TGG CAATG, sense gp160 with start codon underlined) and 635L (TTC CAG GTC TCG AGA TAC TGC TC, antisense). Second-reaction amplification was carried out in 30 cycles at 92°C for 35 seconds, 55°C for 35 seconds, and 68°C for 210 seconds. Ten microliters of the final PCR reaction was analyzed by agarose gel electrophoresis to confirm amplification of the PCR product and the absence of satellite bands. The PCR product was ligated directly into pcDNA3.1-TA plasmid (Invitrogen) per the manufacturer's instructions, and direct automated sequencing of cloned P3gp160 was carried out (Rockefeller University, New York, NY). Sense and antisense strands were sequenced using 10 primers to give at least 2-fold sequencing redundancy. Sequences were aligned using Sequencher 4.1 software (Gene Codes Corporation, Ann Arbor, MI) and translated to the amino acid sequence.
For subcloning of P3gp160 into the 3′ SHIVSF162 genome,16 an expression plasmid for P3gp160 was constructed by replacing a 2.3-kilobase (kb) NsiI/BamH1 fragment of the pCAGGS SF162 plasmid14 with the corresponding sequences of P3gp160. An SphI site immediately 5′ of the ATG start codon of HIV-1 tat and an XhoI site 3′ of the TAA termination codon of HIV-1 env were then introduced into the pCAGGS P3gp160 plasmid, and a 3-kb SphI/XhoI fragment was prepared for substitution into the corresponding region of 3′ SHIVSF162. Infectious molecular clone SHIV P3gp160 was recovered by cotransfection of 293T cells with the 3′ SHIV P3gp160 and the 5′ SIV genomes, followed by cocultivation with CEM × 174 5.25 M7 cells. Stocks of SHIV P3gp160 were propagated and titered in human peripheral blood mononuclear cells (PBMCs).
All infections of adult rhesus macaques (Macaca mulatta) were carried out at the Tulane National Primate Research Center in compliance with the Guide for the Care and Use of Laboratory Animals. All animals were confirmed as serologically negative for simian type D retrovirus, simian immunodeficiency virus (SIV), and simian T-cell lymphotrophic virus before studies. Intravenous and intrarectal inoculations were carried out with 4.2 × 103 50% tissue culture infectious doses (TCID50) of SHIV P3gp160. Four animals were infected by the intravenous route (AL84, DJ39, N570, and P645) and 2 by intrarectal inoculations (AP01 and V127). Whole blood was collected in Vacutainer tubes and was fractionated to PBMCs and plasma, which were frozen until further analysis. Plasma viremia was determined by branched DNA analysis (Bayer Diagnostics, Emeryville, CA), and lymphocyte subsets (CD3+, CD4+, and CD8+) were quantified by TruCount (Becton Dickinson, San Jose, CA).
Cell surface expression of lymphocyte antigens was identified by staining of PBMCs with monoclonal antibodies, followed by flow cytometry using a FACSCalibur (Becton Dickinson Immunocytometry Systems). Analysis was performed using CELLQuest software. The monoclonal antibodies used in this study included anti-monkey CD3-fluorescein isothiocyanate (FITC; clone FN-18, Biosource), anti-human CD4− peridinin chlorophyll protein (PerCP) (clone L200, BD Biosciences), anti-human CD28− allophycocyanin (APCs; clone 28.2, BD Biosciences), and anti-human CD95-phycoerythrin (PE; clone DX2, BD Biosciences). Lymphocytes, initially identified by their forward and side scatter characteristics, were gated based on the expression of CD3 and CD4. The CD3+CD4+ lymphocytes were then analyzed for expression of CD28 and CD95, antigens that differentiate naive (CD28highCD95low) versus memory (CD28high/lowCD95high) T-cell subsets for rhesus macaques.17
SHIVSF162P3 Contains Amino Acid Substitutions in gp120 and gp41
Figure 1 shows a comparison of the amino acid sequence of P3gp160 and SF162 gp160. Fourteen amino acid substitutions in P3gp120 previously described were confirmed, with 8 predicted additional amino acid changes in gp41. These substitutions included E → I and D → G amino acid changes in the N-terminal heptad repeat and C-terminal heptad repeat, respectively, of gp41. In addition, 1 and 2 amino acid substitutions were present in the fusion peptide (FP) and the lentivirus lytic peptide-1 (LLP-1) domain. An SHIV molecular clone expressing the entire P3gp160, designated SHIV P3gp160, was then generated for use in intravenous and intrarectal inoculations of rhesus macaques.
Simian HIV P3gp160 Is Infectious by the Intravenous and Intrarectal Routes and Induces Simian AIDS
Figure 2A shows SHIV P3gp160 replication in animals inoculated by intravenous and intrarectal exposures. Four of 4 and 2 of 2 animals were productively infected by intravenous and intrarectal inoculations, respectively. Peak viral load reached >106 RNA copies/mL in plasma in all 6 macaques, followed by sustained viral replication in 2 animals (AP01 and N570), with postpeak levels ranging from 104 to 106 RNA copies/mL. Transient peripheral CD4+ T-cell loss was seen in 4 of the 6 infected animals (see Fig. 2B), but in the 2 animals with sustained viremia, the CD4+ T-cell counts gradually declined and remained low (≤200 cells/μL of blood) during the course of infection. The spectrum of postacute viremia as well as peripheral CD4+ T cells seen in the SHIV P3gp160-infected macaques is reminiscent of that observed for animals infected with the pathogenic SHIVSF162P3 isolate.13
Analysis of peripheral CD3+CD4+ T cells in macaques AP01 and N570 during peak viremia (3wpi) by 4-color flow cytometry revealed substantial depletion of the memory T-lymphocyte subset (CD28high/low/CD95high: from a baseline of 52.2% to 35.5% in AP01 and from 65.3% to 36.8% in N570) but not the naive T-lymphocyte subset (CD28high/CD95low), a pattern similar to that seen in a representative macaque infected with the pathogenic SHIVSF162P3 isolate (R183; Fig. 3). Macaques AP01 and N570 were euthanized with AIDS-defining symptoms at 34 and 67 weeks after infection, respectively. At the time of death, macaque AP01 had a body weight loss greater than 20%; Mycobacterium avium infection; and severe, chronic, active myocarditis with necrosis and fibrosis. Histologic and pathologic examination indicated hyperplasia of the spleen, lymph nodes, liver, and bone marrow. Postmortem examination of macaque N570 revealed marked atrophy and involution of the thymus, with myeloid and erythroid hypoplasia characteristic of the terminal stages of SIV/SHIV disease. Pneumocystis pneumonia with pulmonary cryptosporidiosis suggested significant immunosuppression in macaque N570. Significant depletion (50%-70% loss) of CD4+ T lymphocytes in the lamina propria (LP) of the intestine of macaques AP01 and N570 was seen at necropsy (Table 1). Cells in the bone marrow and spleen were similarly depleted, whereas lymph node compartments demonstrated a more modest loss (22%-51%). Therefore, infection with SHIV P3gp160 is pathogenic, with preferential targeting of memory T cells (CD95high), which resulted in greater depletion in the gut.
This is the first report of a CCR5-tropic SHIV molecular clone that is mucosally transmissible and pathogenic. Intravenous and intrarectal challenge of a total of 6 animals with SHIV P3gp160 resulted in a diversity of set-point levels of viral replication, which parallels HIV infection in human beings. In the 2 macaques that maintained a viral set point, an irreversible but gradual loss of peripheral CD4+ T cells, depletion of intestinal CD4+ lymphocytes, and induction of simian AIDS were seen.
The maintenance of a viral set point and development of simian AIDS in 2 of 6 SHIV P3gp160 clone-infected macaques differed from the clinical pattern previously described in 4 monkeys infected with the SHIVSF162PC (P3gp120) clone.14 The sample size in these studies was too small to draw any meaningful conclusions on the determinants of pathogenesis of SHIVSF162P3. Nevertheless, several mutations of SHIVSF162P3 gp41 are in domains known to affect envelope glycoprotein functions. Mutations in the FP and heptad repeat domains have been implicated in the efficient fusogenicity of HIV Envs,18,19 whereas changes of the LLP-1 domain attenuate SIV mac239 infection20 and inhibit virion Env incorporation and viral replication in vitro.21 Additionally, the cytoplasmic tail of gp41 has been implicated in mediating the efficient incorporation of Env via interaction with the lentiviral matrix protein (p17).22-24 Further studies with the SHIVSF162PC and SHIV P3gp160 molecular clones in additional animals are required to address the contribution of gp41 to increased pathogenesis of the SHIVSF162P3 isolate.
In summary, we demonstrate that the virulence of SHIVSF162P3 is not a polygenic trait, requiring only adaptive changes in the envelope glycoprotein. Given the importance of R5 viruses in the transmission and establishment of early infection, the derivation of an R5 SHIV molecular clone that models HIV infection in human beings in terms of transmissibility and spectrum of disease induction is of particular relevance. Its use should be of practical value not only in studies of viral pathogenesis and transmission but for the optimization of challenge viruses in vaccine trials.
The authors thank Tina Boadi, Nataliya Trunova, and Lily Tsai for skillful technical and organizational support. Silvana Tasca and Peter Lopez are acknowledged for help with fluorescence-activated cell sorting. The P3gp160 sequence has been deposited in GenBank, accession number AY988107.
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