- Open Access
Role of the long cytoplasmic domain of the SIV Env glycoprotein in early and late stages of infection
© Vzorov et al; licensee BioMed Central Ltd. 2007
Received: 20 September 2007
Accepted: 14 December 2007
Published: 14 December 2007
The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity.
We compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly.
The results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.
The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. There is evidence for intracellular interaction of Env with the matrix protein [1–4], and the Env glycoprotein directly influences the site of release of virus particles in polarized epithelial cells . The cytoplasmic tail of the Env glycoprotein is required for such interactions and has effects on Env incorporation and infectivity [3, 6]. In addition, removal of the cytoplasmic domain can increase the expression of Env on the surface of infected cells, its incorporation into VLPs or membrane vesicles [7–9] and the fusion activity of the Env glycoprotein [10, 11].
SIV and HIV Env glycoproteins contain a relatively long cytoplasmic domain (150–200 amino acids) compared with most other retroviral Env glycoproteins. Nonhuman primates in Africa that are natural hosts for SIV appear to be disease resistant when infected with SIV, whereas nonnatural Asian macaque hosts such as rhesus macaques exhibit progressive CD4+-T-cell depletion and AIDS [12–14]. When SIV strains were passaged on human cell lines they frequently acquired a premature stop codon and expressed a truncated Env glycoprotein that lacks all but approximately 20 amino acids of the cytoplasmic domain [15–18]. However, molecular clones of SIV with truncated Env only establish transient infection in rhesus macaques . Variants with truncated Env are commonly isolated from both types of infected monkeys [15, 17, 19]. However, variants of HIV with truncated Env are rarely isolated from infected patients, even though HIV-1 infected patients can harbor viruses with truncated Env that are able to mediate CD4-independent infection of CD8+ cells .
By budding through lipid rafts in T-cells, HIV and SIV selectively incorporate raft marker proteins and exclude non-raft proteins . The depletion of cholesterol from viral membranes inactivates and permeabilizes HIV and SIV virions . These results indicate a critical role of lipid rafts in the biology of these viruses. It was reported that HIV budding in primary macrophages occurs through the exosome release pathway . A non-pathogenic molecular clone SIVmac1A11 closely related to SIVmac239 but with a truncated Env, which was isolated from an infected rhesus macaque, was able to replicate in monkey macrophages, rhesus PBMC, and human T-cells. However, a pathogenic clone of SIVmac239 was restricted for replication in monkey macrophages and human T-cells [16, 17, 24]. These results indicated that virus replication capacity in different cell lines does not correlate with in vivo virulence.
In the present study we have compared molecularly cloned SIV isolates with sequence differences in the Env glycoprotein, acquired during adaption to human T cells, to investigate the effects of the long cytoplasmic tail of the Env glycoprotein on early steps of replication as well as assembly of SIV. We further compared the replication of these viruses in dividing and non-dividing cells.
Properties of SIV variants
Phenotypic properties of SIV.
Phenotypic properties 1
length of Env CT
sensitivity to neutralization
SIV post-entry replication in dividing vs. non-dividing cells
Taken together, the results indicate that virus entry into cells was similar for SIV with full length or truncated Env in dividing vs. non-dividing cells. The full length Env glycoprotein exhibited a significant effect on the efficiency of SIV postentry replication events compared with truncated Env, but virus with truncated Env can overcome this restriction by high multiplicity of infection.
Production of progeny SIV in dividing and non-dividing cells
Production of Gag antigen SIV in dividing and non-dividing CEMx174 cells.
MTT 1 +aphid1day/+aphid 3 days (OD)
Viability index (fold difference)
p27 ng/ml 2 -aphid.3 days
p27 ng/ml2 +aphid.3 days (x3)3
Production of Gag antigen SIV in macrophages.
Macaque macrophages p27 ng/ml 1
Human macrophages p27 ng/ml 1
Replication of SIV variants generated in human 293T cells.
JC-53B titer b IU c /ml
ELISA (p27) b ng/ml
(fold difference from SIVmac239)
1 × 103
6 × 103
1 × 103
3 × 104
Effects of modifications in the long cytoplasmic tail on lipid raft association and assembly of SIV in 293T cells
To compare the assembly of different Env glycoproteins into virions, we transfected human 293T cells with equal amounts of proviral DNA. At 3 days post transfection cells and supernatants were collected and analyzed by RT assay (not shown). We found similar levels of RT activity in supernatants from cells infected by SIV with full length or truncated Env glycoproteins. The lowest RT activity, about 100-fold lower than in other SIV samples, was observed in supernatants from cells infected by SIV with the C787S Env mutant which eliminated palmitoylation. The infectivity titer of SIV with truncated Env was about 6 to 30-fold higher than SIV with full length Env as described above (Table 4). These results indicate that palmitoylation enhances virus replication and/or assembly viruses with full length Env but is not required in viruses with truncated Env.
Effects of full length and truncated Env on host-cell gene expression
Comparison of mRNA responses by real-time PCR1.
SIVmac239T/SIVmac239 (fold difference)2
The differences in properties between SIV with full length or truncated Env have been previously studied with respect to pathogenicity , fusion activity [10, 11], and assembly [4, 9, 25]. In the present study we had several goals: to study the possible role of the long cytoplasmic tail of the Env glycoprotein in post-entry events, to examine the lipid raft association of Env glycoproteins with full length or truncated cytoplasmic tails, and to compare assembly and release of SIV with full length and truncated Env in dividing and non-dividing cells. We also compared several cloned SIV viruses with sequence differences in the SU and CT subunits of the Env glycoprotein, that were related to adaptation to HUT78 cells .
The early steps of HIV and SIV infection include the attachment of viruses to host cells, entry and transport of the genome to the transcription site, formation of the PIC, and import to the nucleus. Electron microscopic studies showed that HIV cores were disrupted shortly after virus-cell fusion  and viral RNA and associated proteins were released into the cytoplasm and were likely to interact with the cytoskeleton . We found that early steps in replication of SIV with full length or truncated Env were similar in dividing and non-dividing cells. Our results also indicated that internalization of SIV was correlated with amount of p24 input, but not with differences in Env glycoproteins (not shown). Previous studies also indicated that viruses might be internalized into cells irrespectively of CD4 surface expression and with almost equal efficiencies in cells susceptible or not susceptible to HIV infection . The most striking differences were observed when we compared post-entry relocation of SIV with full length or truncated Env using similar input virus levels. The proviral DNA of SIVmac239 with full length Env was transported to the nucleus about 20-fold more efficiently than SIVmac239T with truncated Env, and 100-fold more efficiently than the SIVmac18T variant with a truncated cytoplasmic tail and with a single mutation A239T in the SU subunit. In contrast, the proviral DNA of SIVmac18 with a full length Env and with a single mutation A239T in the SU subunit was transported to nucleus almost as efficiently as the parental SIVmac239. Env glycoproteins are not involved in nuclear import of the HIV pre-integration complex , which may suggest that the effects of Env glycoproteins during early steps of SIV infection is associated with other steps in post-entry replication.
We observed release of infectious SIV particles with full length Env in monkey PBMC cells, but a restriction of particle release in human CEMx174, HUT78, epithelial 293T, or in macrophages. These results are consistent with previous studies indicating that replication of T-tropic SIV and HIV with full length Env is inhibited at a post-nuclear step in macrophages [36, 37]. Our results also demonstrated that a mutation in the long cytoplasmic tail that eliminates palmitoylation did not abolish Env incorporation into lipid rafts as was described for HIV-1 , but prevented virus assembly. In contrast to HIV-1  our results indicate that palmitoylation of the SIV Env cytoplasmic tail is not a prerequisite association with detergent insoluble microdomains. Similar results have been reported for EBV; the interaction of LMP-1 with lipid rafts was shown to be independent of palmitoylation . Furthermore, palmitoylation of viral transmembrane proteins does not necessarily trigger interaction with lipid rafts, since palmitoylated VSV G protein is found in a TX-100 soluble membrane fraction . Palmitoylation was critical for infectivity of SIV with full length Env, and also may impact HIV-1 infectivity [39, 42]. Inhibitory factors such as TRIM5α target the CA and/p2 components of the incoming virus and presumably would be able to restrict infection of both viruses with full length and truncated Env [43, 44].
In contrast to SIV with full length Env, similar levels of assembly and release were observed for SIV with truncated Env in monkey PBMC, human HUT78, CEMx174, 293T, growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We previously observed that SIVmac239T Env with a truncated cytoplasmic tail exhibited the ability to self-associate on the cell surface and assemble into a more closely packed array than full-length Env . Our results indicated that the long cytoplasmic tail of the Env glycoprotein is required for incorporation of Env into lipid rafts, but Env truncation allows SIV to replicate under conditions that are non-permissive for SIV with the full length Env glycoprotein. Since SIV viruses with truncated Env glycoproteins are able to establish productive infection, lipid raft association is apparently not required for virus replication and truncated Env is assembled into infectious SIV virions even though it was not incorporated into lipid rafts. Truncation of the cytoplasmic domain of the SIV Env glycoprotein alters the conformation of the external domain and results in more stable oligomers of TM glycoprotein , and the truncated Env glycoprotein is more fusogenic than the full length Env [10, 11]. These features for incoming virus particles may result in less dependence on the lipid composition of the viral membrane. However, a recent study reported that cholesterol-depleted HIV-1 virions exhibited a defect in internalization . Taken together, the results suggest that SIV with a truncated cytoplasmic tail can overcome a restriction in post-nuclear replication events, but exhibits a defect in early replication events in human and monkey cells.
The present results indicate that a possible basis for defective replication of SIV with truncated Env in primates may be a restriction during an early step of replication, whereas defective replication of SIV with full length Env in human T cells may result from a restriction during a late step of replication and assembly. Comparable host-cell transcriptional responses in rhesus monkey PBMC to both types of virus infection also indicates that cells respond similarly to replication of SIV with full length or truncated Env. A mutation in the Env sequence relating to T cell adaptation alters SIV properties including sensitivity to neutralization, level of Env incorporation, rate of replication and association with lipid rafts during the course of adaptation to human cells.
Cell and virus stocks
The recombinant monkey cell lines sMAGI and human MAGI-R5 were obtained from the NIH AIDS Research and Reference Reagent Program. T-cell line HUT78 and T-B hybrid cell line CEMx174 were obtained from the American Type Culture Collection (Manassas, VA). The recombinant epithelial human cell line JC53-BL (indicator cell line), which is a derivative of HeLa cells that expresses high levels of CD4 and coreceptors CCR5 and CXCR4 , was obtained from Dr. J. Kappes (University of Alabama, Birmingham). The human 239T cell line was kindly provided by Dr. S. L. Lydy. Rhesus monkey PBMCs were separated by centrifugation of whole blood over LSM Lymphocyte Separation Medium (ICN Biomedicals Inc., Costa Mesa, CA). Cells were then stimulated with concanavalin A (Con A, 5 μg/ml in RPMI 1640 containing 10% heat-inactivated fetal calf serum; interleukin-2, human (hIL-2), 10 U/ml; 10 mM HEPES; and antibiotics) for three days before virus infection. To prepare monkey macrophages, PBMC were isolated as described above. Cells (3 × 107 in RPMI 1640 containing 15% human AB+ serum, 1.5 ng/ml of M-CSF, and 0.08 ng/ml of GM-CSF) were seeded into 100-mm plates or split into 24-well plate and incubated for 4 days to allow adherence of monocytes. After removal of nonadherent cells, cells were incubated for another 3–4 days before infection.
SMAGI, MAGI-R5, JC53-BL, and 239T cells were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. HUT78 and CEMx174 cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics, and buffered by 10 mM HEPES.
Preparation of cloned SIV stocks, standardization of virus titers, and conditions for virus infection were done as described earlier . It is commonly accepted to use the infectious titer  or TCID50  for measurement of the quantity of SIV and HIV. However, these methods are not able to precisely compare viruses with different properties such as rate of replication or production of non-infectious particles. We used infectious the index (IU/ng) which is the ratio between infectious titer and core antigen, which is taking both of these characteristics into consideration.
Prior to cell infection, virus preparations were treated with 200 U/ml RNase-free DNase I in growth medium containing 10 mM MgCl2 for 30 min 37°C to remove contaminating proviral DNA . Plasmid pHIVSG3 containing the HIV-1 provirus (SG3) with a deleted env gene was a generous gift from Beatrice Hahn. Plasmid pCMV-GP encoding the Ebola envelope protein GP was provided by C. Yang. The plasmid pRB239ser-787 which carried a mutation in the long cytoplasmic tail of the Env glycoprotein of SIVmac239 C787S to eliminate palmitylation (see below) was digested by NheI and BglII and the resulting fragment with the mutation was introduced in plasmid p3'239 which contained the 3' portion of molecularly cloned SIVmac239  in identical restriction sites. The plasmid, designated p3'239ser-787, was used to obtain a mutant virus as described above.
Construction of recombinant vaccinia viruses
Recombinant vaccinia viruses expressing the SIVmac239-Env or SIVmac239-EnvT were described previously . For the construction of SIVmac239-EnvC787S the codon TGC (cysteine) was changed to AGT (serine) by overlapping PCR. The env gene was amplified from p239SpE3' (NIH AIDS Research and Reference Reagent Program) by using the following primers: primer A (with EcoRI restriction site), CAAAGAATTCAGTATGGGATG; primer B (overlapping primer), GGTTTCTACTGTTGCTGA; primer C (overlapping primer), TCAGCAACAGTAGAACC; and primer D (with restriction site of StuI), GTATTTCTAGGCCTCACAAGAG. Primers B and C carried the codon to be changed. Two PCR amplifications were carried out by using the p239SpE3' plasmid as template. Each PCR was carried out for 25 cycles with steps of 1 min at 95°C, 2 min at 50°C, and 3 min 72°C. The PCR products were purified with a gel extraction kit (Qiagen) according to the manufacturer's protocol. The two overlapping PCR fragments AB and CD were joined by mixing and a PCR reaction with the external primers A and D was performed. The resulting PCR fragment AD was initially cloned in the pDrive vector (Qiagen). The plasmid was digested with EcoRI and StuI and the fragment was cloned in vector pRB21. The resulting plasmid was designated pRB239ser-787 and used for preparation of recombinant vaccinia virus as described .
Conditions for infection with SIV were described previously . At 24 h before infection, 3 × 106 cells were treated with 5 μg/ml aphidicolin, and cells were inoculated with SIV for 2 h in medium with 15 ug/ml DEAE-dextran with or without aphidicolin.
After this incubation unbound virus was removed by three washes and medium with or without aphidicolin was added. For 3 day samples, new medium with 5 μM AZT and with or without aphidicolin was added after 1 day. After 1 and 3 days, the culture supernatant and cells were harvested from each well and used for assays. The p27 content was determined by ELISA Core Antigen assay (Coulter Corporation). The infectivity of virus particles was determined by β-galactosidase assays in JC53-BL , MAGI-R5 or SMAGI cells [29, 59].
Supernatants, cell and nuclear extracts
The supernatants were harvested and clarified by centrifugation at 3.5 k for 20 min (GS-15R, Beckman). Cells were washed three times with PBS and lysed in RIP buffer  and production of Gag antigen was analyzed by SIV Core Antigen Assay (Coulter Corporation). The culture supernatants were also assayed for RT activity by colorimetric reverse transcriptase assay (Roche).
To prepare cell extracts, the cells (3 × 106) were suspended in 0.01 M NaCl, 0.01 M MgCl2 [pH 7.4] for 10 min on ice and then lysed by addition of NP-40 to 1% followed by vortexing as described previously by . Nuclei were recovered by centrifugation at 12,000 × g for 2 min, and nuclear DNA was extracted with a Dneasy Tissue kit (Qiagen) and analyzed by RT-PCR.
RNA preparation and microarray analysis
Total RNA was extracted from cells by using the Rneasy kit (Qiagen, Valencia, CA), according to the manufacturer's protocol. Reverse transcription, second-strand synthesis, and probe generation were accomplished by standard Affymetrix protocols. The Gene Chip HG_U133_Plus 2.0 array (Affymetrix), containing ≈ 33,000 known genes, was hybridized, washed, and scanned according to Affymetrix protocols within the Baylor Affymetrix Core facility. Changes in cellular mRNA levels after SIV infection were compared with mRNA levels in controls that were identically plated, treated, and incubated in the absence of virus. GeneSpring, version 6.2 was used to normalize and scale results and compare viral responses to those of controls. The program clusters increases or decreases of expression levels as the fold change relative to control.
Real-time PCR amplification for SIV
Quantification of proviral DNA from infected cells was performed by real-time PCR using the TaqMan amplification system as described elsewhere . For PCR amplification for the SIV gag region, forward and reverse PCR primers were SIVgagF AGTACGGCTGAGTGAAGGCAGTA and SIVgagR GACCCGCGCCTTTATAGGA, respectively. The fluorogenic SIVgag probe CGGCAGGAACCAACCACGACG was modified with FAM/TAMRA . PCR amplification for the SIV 2LTR region was carried out. Forward and reverse PCR primers were U3U5-2LTRF GGAACGCCCACTTTCTTGATGTATA and U3U5-2LTRR CGGCGGCTAGGAGAGATG. The fluorogenic 2LTR probe was SIV U3U5-2LTRM2 FAM AACACACACTAGCTAATACAG. Nuclear DNA samples corresponding to equal numbers of cells infected by SIV were analyzed in parallel. Fluorescence was recorded as a function of PCR amplification cycle. Quantitative SIV determinations were made by comparison with a standard curve produced by using serial dilution of plasmid DNA with a 1890 bp region of the SIVmac239 gag gene .
RNA isolation and cDNA synthesis
To determine the mRNA transcription profile of selected genes the relative quantitative real-time PCR was performed. PBMC were harvested from cell culture and lysed immediately with 350 μl of lysis buffer from the MagNA Pure LC RNA Isolation Kit III (Tissue) (Roche), then frozen at -80°C. Collected samples were extracted with a MagNa Pure LC – robotic workstation (Roche Molecular Biochemicals) with the same kit using the external lysis protocol. Total RNA was eluted in 60 μl of water and optical density measurements were taken immediately. All total RNA was reverse-transcribed using a High-Capacity cDNA Archive Kit Protocol (Applied Biosystems Inc.).
Quantitative Real-Time PCR Analysis
The reaction was carried out on a 384-well optical plate (Applied Biosystems) in a 20-μl reaction volume containing 30 ng of cDNA per reaction with TaqMan Universal PCR Mastermix, Applied Biosystems. All sequences were amplified using the Applied Biosystems 7900HT Sequence Detection System with the PCR profile: 50° for 2 min, 95°C for 10 min, followed by 45 cycles at 95°C for 15 s, and 60°C for 1 min. Samples were tested in duplicate, in parallel with the housekeeping gene GUSB. For relative quantitation delta-delta Ct analysis was applied to recalculate the fold differences between samples.
Primer and probe sequences
Oligonucleotide primers and probes for IL-2, IL-4, IL-6, TNF-α, IFN-α, TNF-β, and Mx were used as described by . For IL-10 were used two sets of primers and probes. For IL-10 assay a first set of oligonucleotide primers and probe were used as described by  and a second set described below. For other assays oligonucleotide primers and probes were designed using the Primer Express Software (Applied Biosystems) based on published rhesus macaque sequences.
IFNγ : F – GAAAAGCTGACCAATTATTCGGTAA,
R – GCGACAGTTCAGCCATCACTT,
P – 5'FAM – CCAACGCAAAGCAGTACATGAACTCATCC – TAMRA-3';
IL10: F – GTCATCGATTTCTTCCCTGTGAA
R – CTTGGAGCTTACTAAAGGCATTCTTC
P – 5'FAM – CCTGCTCCACGGCCTTGCTCTTG – 3'TAMRA;
IL-12p40: F – TGAAGAAAGACGTTTATGTTGTAGAATTG,
R – TGGTCCAAGGTCCAGGTGAT,
P – 6FAM – CTGGTACCCGGATGC – MGBNFQ
IL-7: F – GATGGCAAACAATATGAGAGTGTTCT,
R – CAATTTCTTTCATGCTGTCCAATAAT,
P – 6FAM – TGGTCAGCATCGATC-MGBNFQ;
IL-15 F – AGCTGGCATTCATGTCTTCATTT,
R – CACCCAGTTGGCTTCTGTTTTAG,
P – 6FAM – CTGTTTCAGTGCAGGGC – MGBNFQ.
Primers and probes were obtained from Applied Biosystems with assay ID as follows: SPIB – Hs00162150_m1; PU.1 – Hs00231368_m1; IRF1 – Hs00971959_m1; IRF2 – Hs00180006_m1; IRF3 – Hs00155574_m1; IRF4 – Hs00277069_m1; IRF5 – Hs00158114_m1; IRF7 – Hs00185375_m1; IFNA1 – Hs00256882_s1. All these assays were designed based on human sequences, and before implementation all assays were validated for rhesus macaques.
Isolation of lipid raft proteins
Radioactively labeled cells expressing different recombinant Env glycoproteins were washed three times with ice cold PBS+++ and then lysed on ice in 750 μl TNE buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA with a protease inhibitor cocktail (Roche)) with 0.5% (v/v) TX-100 for 20 min as described previously . The lysate was passed 10 times through a 25 G needle on ice and subsequently centrifuged at 8000 × g for 10 min at 4°C. The supernatant was brought to 40% sucrose by adding 750 μl of 80% (w/v) sucrose in TNE, loaded into the bottom of a SW41 centrifuge tube and overlaid with 6 ml of 30% (w/v) sucrose in TNE and 3.5 ml 5% (w/v) sucrose in TNE. The samples were spun to equilibrium at 200,000 × g for 13–16 hr. Eleven fractions with volume of each 1 ml were collected started from the top of the gradient and subjected to immunoprecipitation with monkey anti-SIV serum. Samples were analyzed with an SDS-8% PAGE gel and subsequent autoradiography.
For the MTT assay, aphidicolin treated or untreated CEMx174 cells in 96-well plates were infected with SIV. After 24 h or 72 h incubation, 10 μl of MTT (10 mg/ml) reagent was added to 100 μl of medium in each well. After 4 h incubation at 37°C, 100 μl acidic isopropanol (0.04 M HCl in absolute isopropanol) was added. The absorbance was read in a computer-controlled photometer. The absorbance at 690 nm was automatically subtracted from the absorbance at 540 nm to eliminate the effects of non-specific absorption. The MTT assay, which provides an indication of mitochondrial integrity and activity, is not dependent on the cell cycle.
This study was supported by NIH grants AI028147 and AI45883 from the National Institute of Allergy and Infectious Diseases and Emory CFAR grant (P30 AI050409) for using real-time PCR instruments. A.W. was supported by a fellowship from the Bundesministerium für Bildung und Forschung, Germany (BMBF-LPD 9901/8-29).
The authors thank Dahnide Taylor for technical assistance and Erin-Joi Collins for assistance in preparing the manuscript.
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