Organic anion transporting polypeptides in the hepatic uptake of PBDE congeners in mice.

BDE47, BDE99 and BDE153 are the predominant polybrominated diphenyl ether (PBDE) congeners detected in humans and can induce drug metabolizing enzymes in the liver. We have previously demonstrated that several human liver organic anion transporting polypeptides (humans: OATPs; rodents: Oatps) can transport PBDE congeners. Mice are commonly used to study the toxicity of chemicals like the PBDE congeners. However, the mechanism of the hepatic PBDE uptake in mice is not known. Therefore, the purpose of the current study was to test the hypothesis that BDE47, BDE99, and BDE153 are substrates of mouse hepatic Oatps (Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1). We used Human Embryonic Kidney 293 (HEK293) cells transiently expressing individual Oatps and quantified the uptake of BDE47, BDE99, and BDE153. Oatp1a4, Oatp1b2, and Oatp2b1 transported all three PBDE congeners, whereas Oatp1a1 did transport none. Kinetic studies demonstrated that Oatp1a4 and Oatp1b2 transported BDE47 with the greatest affinity, followed by BDE99 and BDE153. In contrast, Oatp2b1 transported all three PBDE congeners with similar affinities. The importance of hepatic Oatps for the liver accumulation of BDE47 was confirmed using Oatp1a4-, and Oatp1b2-null mice.

The flame retardants, polybrominated diphenyl ethers, are pregnane X receptor activators.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and are universally present in the environment. An exponential increase in PBDE concentrations in the U.S. population have been reported over the last 3 decades. PBDEs 47 (tetraBDE) and 99 (pentaBDE) are the most commonly detected PBDE congeners in the environment and in human samples. PBDE209 (decaBDE) is the only remaining PBDE flame retardant commercially manufactured in the United States. Several PBDEs are known to induce cyp3a in rats, but the mechanism of induction remains unclear. The goal of this study was to clarify the mechanism by which PBDE congeners induce cyp3a. Treatment of C57BL6 mice with PBDEs 47, 99, and 209 induced gene expressions of cyp3a11 and 2b10, but not cyp1a1/2. Because the first two genes are known target genes of pregnane X receptor (PXR), a ligand-activated transcription factor in the nuclear hormone receptor superfamily, we hypothesized that PBDE congeners are PXR activators. Using reporter gene luciferase assays, the present data show that PBDEs 47, 99, and 209 activated PXR and its human counterpart, steroid X receptor, but not aryl hydrocarbon receptor. Furthermore, induction of cyp3a11 and 2b10 by PBDEs 47, 99, and 209 was markedly suppressed in PXR-knockout mice, indicating that PBDE congeners activate PXR in vivo. In summary, our study provides the first evidence that PBDEs are activators for xenobiotic nuclear receptor.

APOBEC3G expression is restricted to neurons in the brains of pigtailed macaques.

The Vif protein of human immunodeficiency virus-1 (HIV-1) has been shown to interact with members of the APOBEC family of cytidine deaminases, particularly APOBEC3G/F. In this study, we isolated RNA from 12 regions of the brain from two pigtailed macaques that were exsanguinated and perfused with saline. Our results indicate that APOBEC3G was detected in all regions of the brain analyzed. Immunoblot analysis using lysates prepared from these same regions of the brain and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on whole brain sections. Our results clearly show that the pyramidal neurons within the gray matter of cerebral and cerebellar cortices express APOBEC3G. However, APOBEC3G expression in the pyramidal neurons appeared to be nuclear or associated with nuclei. In contrast to our findings in the cerebral cortex, immunohistochemical analysis of the spleen and kidney tissues revealed that APOBEC3G expression in the cells of these tissues was predominantly cytoplasmic. We further investigated the expression of APOBEC3G in astrocytes. Immunohistochemical staining of serial sections was performed using antibodies to glial fibrillary acidic protein (GFAP) and APOBEC3G. As expected, the cortical and cerebellar white matter showed extensive immunostaining of astrocytes with the antibody against GFAP but a lack of reactivity to the antibody to APOBEC3G. Additionally, Immunoblot analysis of lysates prepared from primary human fetal astrocytes revealed a lack of APOBEC3G expression. Taken together, these results indicate that APOBEC3G expression is restricted to neurons in the brain and that astrocytes and microglia probably do not express this protein or express it at levels undetectable by immunohistochemistry. These finding have implications for the brain as a potential reservoir for Vif-defective viruses.

Interspecies Differences in the Metabolism of a Multiester Prodrug by Carboxylesterases.

The pentaethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA) referred to as C2E5 is being developed as an orally bioavailable radionuclide decorporation agent. The predicted human efficacy obtained in these experimental animals is confounded by interspecies variations of metabolism. Therefore, in the present study, carboxylesterase-mediated metabolism of [(14)C]-C2E5 was compared in the S9 intestinal and hepatic fractions of human, dog, and rat and their respective plasma. Intestinal hydrolysis of C2E5, resulting in the formation of the tetraethyl ester of DTPA (C2E4), was only detected in human and rat. The primary metabolite in human and dog hepatic fractions was C2E4, whereas the predominant species identified in rat hepatic fractions was the triethyl ester (C2E3). Hepatic hydrolysis of C2E5 causes the formation of C2E4 in human, dog, and rat and C2E3 in rat only. Minimal C2E5 hydrolysis was observed in human and dog plasma, whereas in rat plasma C2E5 converted to C2E3 rapidly, followed by slower further metabolism. Both recombinant CES1 and CES2 play roles in C2E5 metabolism. Together, these data suggest that dogs may be the most appropriate species for predicting human C2E5 metabolism, whereas rats might be useful for clarifying the potential toxicity of C2E5 metabolites.

Identification of a region within the cytoplasmic domain of the subtype B Vpu protein of human immunodeficiency virus type 1 (HIV-1) that is responsible for retention in the golgi complex and its absence in the Vpu protein from a subtype C HIV-1.

The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.

Orally administered DTPA di-ethyl ester for decorporation of (241)Am in dogs: Assessment of safety and efficacy in an inhalation-contamination model.

PURPOSE: Currently two injectable products of diethylenetriaminepentaacetic acid (DTPA) are U.S. Food and Drug Administration (FDA)-approved for decorporation of (241)Am; however, an oral product is considered more amenable in a mass casualty situation. The di-ethyl ester of DTPA, named C2E2, is being developed as an oral drug for treatment of internal radionuclide contamination. MATERIALS AND METHODS: Single-dose decorporation efficacy of C2E2 administered 24-h post contamination was determined in beagle dogs using a (241)Am nitrate inhalation contamination model. Single and multiple dose toxicity studies in beagle dogs were performed as part of an initial safety assessment program. In addition, the genotoxic potential of C2E2 was evaluated by the in vitro bacterial reverse mutation Ames test, mammalian cell chromosome aberration cytogenetic assay and an in vivo micronucleus test. RESULTS: Oral administration of C2E2 significantly increased (241)Am elimination over untreated controls and significantly reduced the retention of (241)Am in tissues, especially liver, kidney, lung and bone. Daily dosing of 200 mg/kg/day for 10 days was well tolerated in dogs. C2E2 was found to be neither mutagenic or clastogenic. CONCLUSIONS: The di-ethyl ester of DTPA (C2E2) was shown to effectively enhance the elimination of (241)Am after oral administration in a dog inhalation-contamination model and was well tolerated in toxicity studies.

Vpu: a multifunctional protein that enhances the pathogenesis of human immunodeficiency virus type 1.

The Vpu protein is the smallest of the proteins encoded by human immunodeficiency virus type 1 (HIV-1). This transmembrane protein interacts with the CD4 molecule in the rough endoplasmic reticulum (RER), resulting in its degradation via the proteasome pathway. Vpu also has been shown to enhance virion release from infected cells. While much has been learned about the function of Vpu in cell culture systems, its exact role in HIV-1 pathogenesis is still unknown. This has been primarily due to the lack of a suitable primate model system since vpu is found only in HIV-1 and simian immunodeficiency viruses isolated from chimpanzees (SIVcpz), and three species of old world monkeys within the genus Cercopithecus. Several laboratories have developed pathogenic molecular clones of simian-human immunodeficiency virus (SHIV) in which the tat, rev, vpu and env genes of HIV-1 are expressed in the genetic background of SIV. The availability of such clones has allowed investigators to assess the role of Vpu in pathogenesis using a relevant animal model. This review will focus on the current understanding of the structure-function relationships of Vpu protein and recent advances using the SHIV model to assess the role of Vpu in HIV-1 pathogenesis.

The primary phase of infection by pathogenic simian-human immunodeficiency virus results in disruption of the blood-brain barrier.

Using the simian-human immunodeficiency virus (SHIV), we have investigated whether the blood-brain barrier (BBB) is compromised during the early stages of infection. Five macaques were inoculated with pathogenic SHIV(50OLNV) for 2 weeks at which time macaques were anesthetized, perfused with saline, and sacrificed. The brains were removed and examined for the disruption of the blood-brain barrier by immunohistochemical staining for the plasma protein fibrinogen in the neural parenchyma. Our results indicate a disruption of the BBB in the five of five macaques inoculated with SHIV(50OLNV) for 2 weeks. Zonula occludens 1 (ZO-1), which is a marker for the tight junctions formed by brain vascular endothelial cells, was largely absent in areas that showed fibrinogen deposition in all five macaques. To determine if the BBB integrity correlated with the initial stages of infection, the brains from two macaques were analyzed that had progressed to end-stage disease following inoculation with pathogenic SHIV(50OLNV) but developed no neuropathology and from two macaques that were inoculated with a gene-deleted, nonpathogenic virus (novpuSHIV(KU-1bMC33)) for over 1 year. Our results indicate that unlike the macaques sacrificed during the acute phase of infection, immunohistochemical staining for fibrinogen in the neural parenchyma was negative and ZO-1 staining was readily detected in the endothelial cells of the blood vessels. The results of this study indicate that the transient loss of BBB integrity is a function of the high level of virus replication that occurs during the acute phase of infection and provides important information on the early stages of lentivirus neuroinvasion.

Infection of human astrocytoma cells with simian-human immunodeficiency virus results in up-regulation of gene expression and altered growth properties.

Recent reports of human immunodeficiency virus-1 (HIV-1) infection of astrocytes suggest a role for astrocytes in HIV encephalitis. In this study, we infected a human astrocytoma cell line with a pathogenic simian HIV (SHIV(50OLNV)) and examined growth patterns and immunomodulatory genes. Approximately 1% of uninfected cells in culture expressed glial fibrillary acid protein (GFAP) whereas 40% of the cells expressed GFAP at 7 days post-inoculation along altered growth patterns. Using targeted cytokine cDNA arrays, we found that SHIV(50OLNV) infection resulted in the up-regulation of several genes including metalloproteinase bone morphogenic protein 1 and chemokines monocyte chemoattractant protein 1 and stromal cell derived factor 1alpha. These data suggest that astrocytic activation, altered morphology and up-regulation of immunomodulatory genes in response to SHIV infection may participate in initiation of inflammation and trafficking of infected monocytes/macrophages into the central nervous system, potentiating the development of HIV encephalitis.

Nonaqueous gel for the transdermal delivery of a DTPA penta-ethyl ester prodrug.

Diethylenetriamine pentaacetic acid penta-ethyl ester, designated as C2E5, was successfully incorporated into a nonaqueous gel for transdermal delivery. The thermal and rheological properties of a formulation containing 40% C2E5, 20% ethyl cellulose, and 40% Miglyol 840® prepared using the solvent evaporation method demonstrated that the gel had acceptable content uniformity and flow properties. In vitro studies showed that C2E5 was steadily released from the gel at a rate suitable for transdermal delivery. Topical application of the gel at a 200 mg C2E5/kg dose level in rats achieved significantly higher plasma exposures of several active metabolites compared with neat C2E5 oil at the same dose level. The results suggest that transdermal delivery of a chelator prodrug is an effective radionuclide decorporation strategy by delivering chelators to the circulation with a pharmacokinetic profile that is more consistent with the biokinetic profile of transuranic elements in contaminated individuals.

Mechanism of polybrominated diphenyl ether uptake into the liver: PBDE congeners are substrates of human hepatic OATP transporters.

Polybrominated diphenyl ethers (PBDEs) are flame-retardants that upon chronic exposure enter the liver where they are biotransformed to potentially toxic metabolites. The mechanism by which PBDEs enter the liver is not known. However, due to their large molecular weights (MWs approximately 485 to 1000 Da), they cannot enter hepatocytes by simple diffusion. Organic anion-transporting polypeptides (OATPs) are responsible for hepatic uptake of a variety of amphipathic compounds of MWs larger than 350 Da. Therefore, in the present study, Chinese hamster ovary cell lines expressing OATP1B1, OATP1B3, and OATP2B1 were used to test the hypothesis that OATPs expressed in human hepatocytes would be responsible for the uptake of PBDE congeners 47, 99, and 153. The results demonstrated that PBDE congeners inhibited OATP1B1- and OATP1B3-mediated uptake of estradiol-17-beta-glucuronide as well as OATP2B1-mediated uptake of estrone-3-sulfate in a concentration-dependent manner. Direct uptake studies confirmed that all three PBDE congeners are substrates for the three tested hepatic OATPs. Detailed kinetic analysis revealed that OATP1B1 transported 2,2',4,4'-tetrabromodiphenyl ether (BDE47) with the highest affinity (K(m) = 0.31 microM) followed by 2,2',4,4',5-pentabromodiphenyl ether (BDE99) (K(m) = 0.91 microM) and 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE153) (K(m) = 1.91 microM). For OATP1B3, the order was the same (BDE47: K(m) = 0.41 microM; BDE99: K(m) = 0.70 microM; BDE153: K(m) = 1.66 microM), while OATP2B1 transported all three congeners with similar affinities (BDE47: K(m) = 0.81 microM; BDE99: K(m) = 0.87 microM; BDE153: K(m) = 0.65 microM). These results clearly suggest that uptake of PBDEs via these OATPs is a mechanism responsible for liver-specific accumulation of PBDEs.

Modulation of the severe CD4+ T-cell loss caused by a pathogenic simian-human immunodeficiency virus by replacement of the subtype B vpu with the vpu from a subtype C HIV-1 clinical isolate.

Previously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIV(SCVpu), in which the subtype B vpu gene from the pathogenic SHIV(KU-1bMC33) was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIV(SCVpu) replicated with slightly reduced kinetics when compared with the parental SHIV(KU-1bMC33) and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIV(KU-1bMC33). To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIV(SCVpu) and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIV(SCVpu) caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIV(KU-1bMC33). These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model.

A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV(KU-1bMC33)) susceptible to rimantadine.

Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV(KU-1bMC33) in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV(M2)) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV(KU-1bMC33)) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV(VpuA19H) replicated with similar kinetics as the parental SHIV(KU-1bMC33) and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV(KU-1bMC33). This SHIV(VpuA19H) virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV(M2). Electron microscopic examination of SHIV(VpuA19H)-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV(M2)-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence that drugs targeting the Vpu TM/ion channel can be effective anti-HIV-1 drugs.

Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIVKU1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques.

The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.

Vpu-mediated CD4 down-regulation and degradation is conserved among highly divergent SIV(cpz) strains.

Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV(cpz)) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV(cpz) isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV(cpz) Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site (SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV(cpz) ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu (SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV(cpz) isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.

Scrambling of the amino acids within the transmembrane domain of Vpu results in a simian-human immunodeficiency virus (SHIVTM) that is less pathogenic for pig-tailed macaques.

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.

Pathogenic and nef-interrupted simian-human immunodeficiency viruses traffic to the macaque CNS and cause astrocytosis early after inoculation.

Several studies have shown that deletion of the nef gene of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) results in attenuated viruses. However, studies have not critically examined trafficking of attenuated viruses to the central nervous system (CNS) at early stages after inoculation. In this study, we investigated the colocalization of pathogenic and vpu-negative, nef-interrupted SHIVs at early stages following inoculation. The first virus, designated SHIV(50OLNV), was isolated from the lymph node of a pig-tailed macaque which developed severe CD4+ T cell loss and neurological disease. The second virus was a molecularly cloned virus in which the vpu gene was deleted and the gene for the enhanced green fluorescent protein from the jellyfish Aequoria victora had been inserted in-frame within the nef gene of the pathogenic SHIV(KU-1bMC33) (designated SHIV(KU-1bEGFP)). Three pig-tailed macaques were inoculated intravenously with equivalent amounts of two viruses, two macaques were inoculated with SHIV(KU-1bEGFP), and two macaques were inoculated with SHIV(50OLNV). The peripheral blood mononuclear cells (PBMCs) were isolated from bleeds obtained 3, 7, 10, and 14 days postinoculation and monitored for syncytia-inducing virus and for fluorescent cells. Virus was detected in the PBMCs as early as 3 days postinoculation and was present throughout the course of this short-term study. At 14 days postinoculation, the macaques were sacrificed and examined for virus in lymphoid tissues and different regions of the CNS following necropsy. Our results revealed the presence of both viruses in lymphoid and CNS tissues, although SHIV(50OLNV) was present to a much greater extent. Histological examination revealed that one macaque displayed signs of meningitis and all three macaques developed massive cortical astrocyte activation as demonstrated by immunostaining for glial fibrillary acidic protein, but only limited microglial activation. In the two macaques inoculated with SHIV(50OLNV), astrocyte activation similar to that in the macaques inoculated with both viruses was observed while no astrocyte activation was observed in macaques inoculated with SHIV(KU-1bEGFP). Thus, this study demonstrates that SHIVs with an intact nef(SHIV(50OLNV)) as well as those lacking a vpu gene and with a nonfunctional nef gene (SHIV(KU-1bEGFP)) are capable of invading the CNS and that pathogenic SHIVs are capable of causing reactive astrocytosis early after inoculation.

Deletion of the vpu sequences prior to the env in a simian-human immunodeficiency virus results in enhanced Env precursor synthesis but is less pathogenic for pig-tailed macaques.

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) has been reported to enhance virion release from infected cells and to down-regulate the expression of CD4 on infected cells. Previous studies have shown that Vpu and the envelope glycoprotein precursor (gp160) are translated from different reading frames of the same bicistronic messenger RNA (mRNA). In order to assess the effect of the Vpu sequences 5' to the Env open reading frame on Env biosynthesis and pathogenesis, we have constructed a deletion mutant of a molecularly cloned chimeric simian--human immunodeficiency virus (SHIV(KU-1bMC33)) in which the entire coding region of vpu upstream of env had been deleted (novpuSHIV(KU-1bMC33)). While both SHIV(KU-1bMC33) and novpuSHIV(KU-1bMC33) synthesized comparable amounts of env mRNA in infected cells, the novpuSHIV(KU-1bMC33)-infected cells synthesized more Env precursor when standardized against the p57 Gag precursor protein. While more Env was synthesized than Gag in novpuSHIV(KU-1bMC33)-infected cells, pulse--chase analysis revealed that p27 Gag protein was released from infected cells with delayed kinetics, a reflection of the lack of a Vpu protein. Inoculation of novpuSHIV(KU-1bMC33) into two pig-tailed macaques resulted in no loss of circulating CD4(+) T cells. However, replicating virus could be detected in the lymphoid tissues (lymph nodes, spleen, thymus) 1 year after inoculation and the thymus of one of the macaques exhibited severe atrophy. The results of these studies indicate that the Vpu coding sequences upstream of Env may attenuate the level of Env precursor biosynthesis but significantly contribute to the pathogenesis of this SHIV in pig-tailed macaques.

The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4(+) T cell loss caused by the simian-human immunodeficiency virus SHIV(KU-lbMC33) in pig-tailed macaques.

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.

Fusion of the upstream vpu sequences to the env of simian human immunodeficiency virus (SHIV(KU-1bMC33)) results in the synthesis of two envelope precursor proteins, increased numbers of virus particles associated with the cell surface and is pathogenic for pig-tailed macaques.

Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5' to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV(Vpenv)) in which a single nucleotide was removed at the vpu-env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV(Vpenv)-infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV(Vpenv)-inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV(KU-1bMC33). Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV(Vpenv)-infected cells compared to cultures inoculated with parental SHIV(KU-1bMC33). Furthermore, virus was observed maturing into intracellular vesicles of SHIV(Vpenv)-infected cells. To assess the pathogenicity of SHIV(Vpenv), three pig-tailed macaques were inoculated with the SHIV(Vpenv) and monitored for 6 months for CD4(+) T cell levels, viral loads, and the stability of the deletion at the vpu-env junction. Our results indicated that SHIV(Vpenv) caused a severe CD4(+) T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4(+) T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV(Vpenv) with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.