Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Mouse models of human T lymphotropic virus type-1-associated adult T-cell leukemia/lymphoma.

Human T-lymphotropic virus type-1 (HTLV-1), the first human retrovirus discovered, is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1-associated diseases has been problematic. Mechanisms of early infection and cell-to-cell transmission can be studied in rabbits and nonhuman primates, but lesion development and reagents are limited in these species. The mouse provides a cost-effective, highly reproducible model in which to study factors related to lymphoma development and the preclinical efficacy of potential therapies against ATL. The ability to manipulate transgenic mice has provided important insight into viral genes responsible for lymphocyte transformation. Expansion of various strains of immunodeficient mice has accelerated the testing of drugs and targeted therapy against ATL. This review compares various mouse models to illustrate recent advances in the understanding of HTLV-1-associated ATL development and how improvements in these models are critical to the future development of targeted therapies against this aggressive T-cell lymphoma.

The human T-cell leukemia virus type 1 p13II protein: effects on mitochondrial function and cell growth.

p13(II) of human T-cell leukemia virus type 1 (HTLV-1) is an 87-amino-acid protein that is targeted to the inner mitochondrial membrane. p13(II) alters mitochondrial membrane permeability, producing a rapid, membrane potential-dependent influx of K(+). These changes result in increased mitochondrial matrix volume and fragmentation and may lead to depolarization and alterations in mitochondrial Ca(2+) uptake/retention capacity. At the cellular level, p13(II) has been found to interfere with cell proliferation and transformation and to promote apoptosis induced by ceramide and Fas ligand. Assays carried out in T cells (the major targets of HTLV-1 infection in vivo) demonstrate that p13(II)-mediated sensitization to Fas ligand-induced apoptosis can be blocked by an inhibitor of Ras farnesylation, thus implicating Ras signaling as a downstream target of p13(II) function.

Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma.

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy seen in the majority of adult T-cell leukemia/lymphoma (ATLL) patients with human T-cell lymphotropic virus type-1 (HTLV-1) infection. HTLV-1 Tax has been shown to complex with ETS-1 and SP1 to transactivate the PTHrP P3 promoter. Previously, we established a SCID/bg mouse model of human ATL with RV-ATL cells and showed that PTHrP expression was independent of Tax. In this study, we report an inverse correlation of PTHrP with tax/rex mRNA in multiple HTLV-1-positive cell lines and RV-ATL cells. Stimulation of Jurkat T cells with PMA/ionomycin upregulated the PTHrP P3 promoter by a previously characterized Ets binding site and also induced protein/DNA complex formation identical to that observed in RV-ATL cells. Further, we provide evidence that cotransfection with Ets-1 and constitutively active Mek-1 in HTLV-1-negative transformed T cells with stimulation by PMA/ionomycin not only resulted in a robust induction of PTHrP P3 but also formed a complex with ETS-1/P3 EBS similar to that in ATLL cells. Our data demonstrate that transcriptional regulation of PTHrP in ATLL cells can be controlled by T-cell receptor signaling and the ETS and MAPK ERK pathway in a Tax-independent manner.

Experimental coinduction of type D retrovirus-associated pulmonary carcinoma and lentivirus-associated lymphoid interstitial pneumonia in lambs.

Two retrovirus-associated pulmonary diseases of sheep [ovine pulmonary carcinoma (OPC); sheep pulmonary adenomatosis], a bronchoalveolar carcinoma, and lymphoid interstitial pneumonia (LIP) were induced simultaneously in 9 of 9 neonatal lambs. The lambs were killed 8-28 weeks after intratracheal injection of lung tumor homogenate or lung fluid derived from sheep with naturally occurring OPC and ovine lentivirus (OvLV) infection. The inoculated lambs developed multifocal neoplasms of alveolar type II cells or nonciliated bronchiolar epithelial cells, LIP, and pulmonary lymph node hyperplasia, and all produced antibody to OvLV. OvLV was isolated from 6 to 7 lambs tested, and infectious center assay of pulmonary lavage cells from 3 lambs revealed that approximately 1 in 1,000 pulmonary lavage cells contained infectious lentivirus. Neither contact control lambs nor control lambs that received ultrafiltered lung fluid developed evidence of either disease or of OvLV infection. Lung fluid or tumor tissue of lambs with OPC contained a 26,000-dalton protein that cross-reacted with antiserum to p27 to Mason-Pfizer monkey virus, a type D retrovirus. The fact that no antigenic cross-reaction between OvLV and type D retroviruses has been demonstrated supports the presence of two retroviruses in sheep with OPC. Although the contributions of each agent to oncogenesis in this model are difficult to evaluate, the rapid development of two retrovirus-induced pulmonary diseases in experimentally inoculated lambs suggests an etiologic or pathogenetic synergism between these two members of the family Retroviridae.

Glycosylation-dependent peptide antigenic determinants of env gp46 HTLV-1.

Human T-lymphotropic virus type 1 (HTLV-I) is a retrovirus associated with adult T-cell leukemia/lymphoma, and the virus infection constitutes a growing public health problem. In a continuing effort to engineer conformationally dependent HTLV-I epitopes that elicit a protective immune response, we have examined the role and functional importance of carbohydrate moieties in specific immune recognition and antibody responses. There have been several reports of the importance of N-linked virus glycosylation in the formation of neutralizing antibodies. Residues 230-257 is predicted to encode two beta-turn/loop regions at 240-244 (LYGPN), 248-257 (VPSSSSTPL) and a glycosylation site at N-244 (NVS). We have successfully engineered and synthesized the 233-253 sequence of gp46 of HTLV-1 with and without GlcNAC at Asn244. Circular dichroism spectroscopy and proton NMR showed the presence of beta-turn conformation in both peptide constructs. Chimeras of the glycosylated and non-glycosylated epitope with promiscuous T-cell epitope were synthesized and shown to elicit high titered antibodies in rabbits specific for the immunogen (SC1MVF and SC2MVF) and the B cell epitope 233-253. Additionally, antibodies to the glycosylated form of the peptide recognized the HTLV-I envelope precursor in radioimmunoassay precipitation assay and react with HTLV-I whole virus preparations in ELISA.

Sexual transmission of simian T-lymphotropic virus type I: a model of human T-lymphotropic virus type I infection.

Simian T-lymphotropic virus type-I (STLV-I) seronegative females placed together with seropositive males for breeding purposes were followed from 1984-1990 to determined seroconversion rates by enzyme immunoassay and western immunoblot analysis. Two of 26 females and 1 of 4 males previously negative for antibodies to STLV-I seroconverted during the study period. Statistical analysis of sexual encounters indicated that the probability of a seronegative female testing positive for STLV-I after a sexual encounter with a seropositive male is less than 4%. These data indicate that even though sexual contact is important in the transmission of STLV-I, it may not be an efficient mode of viral infection. These data also suggest that female-to-male transmission of STLV-I occurs, as recently reported for human T-lymphotropic virus type-I (HTLV-I) infection. These results are important because HTLV-I and STLV-I share many features in common including routes of viral transmission. In addition, the difficulty of clearly quantitating the risk of sexual transmission in humans makes the primate animal model a valuable alternative to study the human infection.

Differential replication and pathogenic effects of HIV-1 and HIV-2 in Macaca nemestrina.

OBJECTIVE: HIV-1 and HIV-2 isolates representing various geographic regions and distinct viral subtypes were examined for their ability to establish both in vitro and in vivo productive infections of Macaca nemestrina (pigtail macaque) peripheral blood mononuclear cells. METHODS: Animals were inoculated with either autologous cell-associated or cell-free viral preparations of selected isolates. HIV-specific immune responsiveness, hematologic changes, genetic variation, and virus burden were monitored as delineators of HIV pathogenesis. RESULTS: HIV-2 replication in vitro and in vivo correlated with nascent antigen production and rising viral titers as determined by infectious center assays. Infection was detectable by polymerase chain reaction amplification of proviral sequences in macaque cells as early as 1 week postinoculation. Two distinct patterns of CD4+ cell depletion induced by HIV-2 infection were observed during the first month postinoculation and characterized by a moderate loss sustained through 20 weeks postinoculation or a substantial loss maintained long-term (> 90 weeks). Identity between inoculating viral stocks and subsequent viral isolates from animals was established comparatively by limited sequence analysis of specific domains within the HIV-2 pol and env genes. In contrast, replication of HIV-1 isolates was limited or only semipermissive in vitro. Intravenous inoculation of HIV-1 field isolates, using conditions successful for HIV-2 (for example, identical viral titers), failed to establish a productive viral infection leading to seroconversion of fluctuations in hematologic cell markers. Infection with a high-titer inoculum of a laboratory-adapted HIV-1 strain in vivo, as demonstrated by polymerase chain reaction analysis, produced seroconversion in the absence of overt viral replication or hematologic variations in one out of four animals. CONCLUSIONS: This system provides for multifaceted modeling of HIV pathogenesis, primarily with HIV-2 and potentially with HIV-1/-2 chimerics, in support of immunotherapeutic developments and critical evaluation of intervention practices.

HTLV-I-associated myelopathy endemic in Texas-born residents and isolation of virus from CSF cells.

We report three Texas-born patients with spastic paraparesis and well-documented infection with HTLV-I. CSF examination showed moderate pleocytosis, protein elevation, and elevated IgG index. Oligoclonal bands were present in two patients. On MRI, one patient had frontal lobe lesions that were low intensity on T1- and high intensity on T2-weighted images. HTLV-I immunoblot studies of serum and CSF revealed reactivity to p19, p24, p53, gp46, or gp68 from all three patients. Titration studies of serum and CSF antibodies on ELISA and immunoblot assays indicated an intrathecal virus-specific response. HTLV-I-specific p19 antigen capture assay and polymerase chain reaction (PCR) demonstrated HTLV-I in lymphocyte cultures derived from each patient's peripheral blood mononuclear cells (PBMC) or CSF cells. Using HTLV-I- and HTLV-II-specific pol and gag primers, PCR studies of PBMC cells obtained directly from the patients demonstrated that the patients were infected with HTLV-I and not HTLV-II. These three cases are to our knowledge the only US cases in whom virus isolation from the CSF has been accomplished. Importantly, two patients may be the first US cases of myelopathy arising from endemic infection.

HTLV-I-associated myelopathy associated with blood transfusion in the United States: epidemiologic and molecular evidence linking donor and recipient.

Six months after receiving 58 units of blood components, a 65-year-old white man from New York City, with no other risk factors for human T-lymphotropic virus type I (HTLV-I) infection, developed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Investigation of blood donors identified a 25-year-old white Hispanic woman from Florida whose platelets had been given to the patient and who was seropositive for the virus on a serum specimen obtained 2 years after the donation. She was born in Cuba and had had 2 sexual relationships with men who either had been born in or had resided in the Caribbean. Polymerase chain reaction (PCR) studies of peripheral blood mononuclear cells indicated that both donor and recipient were infected with HTLV-I. Molecular studies of a 595-nucleotide sequence in the 5' envelope region of HTLV-I indicated that the viruses from donor and recipient were identical in each of 32 positions in which published HTLV-I sequences demonstrate molecular heterogeneity; the donor and recipient viruses were also identical in 2 additional positions in which they differed from all published sequences. Transfusion-associated HAM/TSP has occurred in the United States, but additional cases should be prevented by screening blood donations for HTLV-I. Molecular studies of HTLV-I may prove useful in defining the genetic heterogeneity of HTLV-I isolates in the United States and in studying transmission of this virus.

Soluble recombinant HTLV-1 surface glycoprotein competitively inhibits syncytia formation and viral infection of cells.

Efficient entry into, and infection of, human cells by human T-cell leukaemia virus type-1 (HTLV-1) is mediated by the viral envelope glycoproteins, gp46 and gp21. The gp46 surface glycoprotein binds to an as yet unidentified cell surface receptor, thereby, allowing the gp21 transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral penetration and entry into the host cell cannot occur. The envelope glycoproteins are also a major target for neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and represent ideal constituents for a recombinant HTLV-1 vaccine. Given the importance of the envelope proteins in HTLV-1 pathogenesis there is increasing interest in obtaining sufficient quantities of these proteins for biochemical, biophysical and biological analyses. We have now developed a system for production of large amounts of a glycosylated and functional form of soluble recombinant gp46 (sRgp46), and have used this recombinant material for analysis of envelope function and receptor binding activity. We find that, the sRgp46 molecules expressed in our system are immunologically indistinguishable from the native virally expressed surface glycoproteins; that sRgp46 binds to T-cells in a dose dependent and saturable manner; and that cell surface binding by sRgp46 can be inhibited by neutralising antibodies. Importantly, we demonstrate that these sRgp46 molecules potently inhibit syncytia formation and viral infection of target cells, and that regions outwith the SU domain of envelope are not required for binding to target cells or for inhibiting membrane fusion. The sRgp46 produced in our study will provide new opportunities to investigate envelope-receptor interactions, and will be of utility in defining the conformationally sensitive antigenic determinants of the HTLV-1 surface glycoprotein.

Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB.

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.

Characterization of a B-cell immunodominant epitope of human T-lymphotropic virus type 1 (HTLV-I) envelope gp46.

The immune response elicited by a synthetic peptide derived from an immunodominant external envelope region (Env-5, amino acids 242-257) of human T-lymphotropic virus type 1 (HTLV-I) was tested in a rabbit model of HTLV-I infection. The synthetic peptide elicited a strong antibody response to the HTLV-I envelope protein gp46; however, these antibodies failed to inhibit HTLV-I-mediated cell fusion. Immunized rabbits were not protected from HTLV-I infection as determined by seroconversion to viral core proteins by immunoblot, HTLV-I p24 antigen detection in lymphocyte cultures and polymerase chain reaction for the HTLV-I provirus in lymphocyte DNA. Env-5 peptide immunization failed to induce T-cell lymphocyte proliferative responses in rabbits, but induced antibody responses in T-cell deficient Balb c nu/nu mice suggesting that the antigenic determinant represented by the Env-5 peptide is primarily a B-cell epitope. These results further define an immunodominant epitope of the HTLV-I envelope protein and suggest that potential synthetic peptide vaccines against HTLV-I infection must contain multiple antigens that induce both humoral and cellular immune reactivity.

In vitro cellular tropism of human T cell leukemia virus type 2.

Human T cell leukemia virus type 1 (HTLV-1) and 2 (HTLV-2) are distinct oncogenic retroviruses that infect several cell types, but display their biologic/pathogenic activity only in T lymphocytes. HTLV-1 is associated with adult T cell leukemia, a malignancy of mature CD4(+) T cells, and a chronic neurological disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 is less pathogenic and has been associated with a few cases of a variant of hairy cell leukemia and neurological disease. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4(+) T cells, whereas HTLV-2 in vivo tropism is less clear, but appears to favor CD8(+) T cells. The molecular mechanism that determines the cellular tropism of HTLV-1 and HTLV-2 has not been precisely determined. However, one study by our group has provided evidence that HTLV-1-enhanced viral transcription in CD4(+) T cells may be responsible for its tropism. In an effort to understand HTLV-2 tropism we tested the ability of HTLV-2 to infect, replicate in, and transform purified CD4(+) or CD8(+) T cells in cell culture. After cocultures of purified primary human CD4(+) and CD8(+) T cells with an HTLV-2-producer cell line we measured viral transcription by reverse transcription PCR analysis, virus production by p19(gag) ELISA, proviral integration by DNA slot-blot analysis, surface phenotype by FACS analysis, and cellular transformation. We also measured HTLV-2 long terminal repeat-directed transcription in the presence and absence of Tax in purified CD4(+) and CD8(+) T cells, using transient transfection assays. Our data indicate that CD4(+) and CD8(+) T cells are equally susceptible to HTLV-2 infection. We observed no significant difference in viral transcription based on mRNA and virus production in CD4(+) and CD8(+) T cell cocultures. Although LTR transcription was enhanced 12- to 16-fold in the presence of Tax, there was no significant difference in CD4(+) or CD8(+) T cells. Interestingly, we show that HTLV-2 preferentially transforms CD8(+) T cells in culture. Together, our data indicate that, unlike HTLV-1, HTLV-2 cell tropism is not due to inhibition of viral infection and inefficient gene expression in CD4(+) versus CD8(+) T cells, and likely involves unique interactions with viral and CD8(+) T cell-specific proteins.

SIV, STLV-I and type D retrovirus antibodies in captive rhesus macaques and immunoblot reactivity to SIV p27 in human and rhesus monkey sera.

The prevalence of simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus type 1 (STLV-I), and type D retrovirus (SRV-D) antibodies was determined for 1229 rhesus monkeys (Macaca mulatta) from two research colonies. Serum samples were tested by using enzyme-linked immunosorbent assay (ELISA), immunoblot (IB), and radioimmunoprecipitation assay (RIPA). Seropositive results for the three retroviruses tested were 0 for SIV, 270 (22%) for STLV-I, and 103 (8.4%) for type D retrovirus. Of the rhesus monkey sera, 61 (5.0%) were reactive to SIV gag p27 only, when tested by IB, but were negative when further tested by RIPA. Virus isolation was attempted from cultured peripheral blood mononuclear cells of 35 monkeys whose sera contained only p27 reactivity and none were positive by reverse transcriptase and core antigen assays to detect SIV. No overt clinical signs of immunodeficiency disease or unexplained deaths were evident in either monkey colony. Additionally, 63 of 165 (38%) human sera from various groups (primate center workers, normal donors, health care workers) had weak to moderate IB reactivity only to SIV p27, but 31 of 31 sera tested were negative by RIPA. These sera remained reactive to SIV p27 following absorption with an uninfected cell lysate, after blocking IB strips with various blocking solutions and were reactive to different SIV antigen preparations while remaining negative to human immunodeficiency virus type 1 (HIV-1) by IB and negative to HIV-2 by ELISA. These data underscore the need to adopt criteria for a positive SIV serologic test requiring reactivity against more than one viral gene product. These results also illustrate a potential problem in the testing of human sera for antibodies against simian retroviruses and demonstrate the need for caution in the interpretation of immunoblot results.

In vitro and in vivo functional analysis of human T cell lymphotropic virus type 1 pX open reading frames I and II.

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.

Detection of simian immunodeficiency virus and human immunodeficiency virus type 2 capsid antigens by a monoclonal antibody-based antigen capture assay.

We have tested the ability of a monoclonal antibody-based simian immunodeficiency virus (SIV) p27 capsid antigen assay to detect SIV antigen in supernatants from a variety of infected cell cultures. The antigen capture assay has a sensitivity of approximately 30 pg of SIV p27 capsid antigen/ml. The assay detected SIV p27 capsid antigen in cell culture supernatants from all six strains tested, detected the replication of SIV following the inoculation of the virus in peripheral blood mononuclear cell cultures earlier compared to reverse transcriptase assay, and was more sensitive in detection of the SIV antigen compared to human immunodeficiency virus type 1 (HIV-1) antigen capture assays. The SIV antigen capture assay was used to detect SIV antigen from serum samples and tissue cultures from eight of eight SIVB670-infected rhesus macaques (Macaca mulatta). Similar samples from four control rhesus macaques were negative when tested by the assay. The SIV antigen was detected in virus-infected monkeys during early time periods following inoculation (1 to 3 weeks) or during episodes of CD4+ lymphocytopenia and clinically evident disease. In addition, the SIV antigen capture assay positively identified each of three different HIV-2 strains in cell culture supernatants. The SIV antigen capture assay provides a sensitive and specific method to monitor SIV and HIV-2 capsid antigen in cell cultures and from infected animals. The assay will be an important tool in the utilization of SIV and HIV-2 primate models for HIV-induced acquired immunodeficiency disease syndrome.

Characterization of a HTLV-I-infected cell line derived from a patient with adult T-cell leukemia with stable co-expression of CD4 and CD8.

A long-term T-cell line, termed SP+, was developed from a human T-cell leukemia virus type I (HTLV-I)-infected patient with adult T-cell leukemia that is dependent on exogenous IL-2 for growth. The SP+ expresses a full complimentation of HTLV-I-specific viral proteins, and contains replication competent viral particles. Restriction enzyme digestion followed by Southern blot analysis demonstrated the presence of a single integrated proviral copy and limiting dilution analysis confirmed the clonality of the cell line. Interestingly, phenotypically, the SP+ cell line is CD2+, CD3+ and coexpresses CD4 and CD8, yet lacks TCR alpha beta and TCR tau delta expression. Further ontogenetic characterization of the SP+ cell line demonstrated the lack of thymic T-cell precursor markers, including absence of cell surface expression of CD1, intracellular thymic terminal deoxynucleotidyl transferase (TdT) enzyme, as well as message expression for V(D)J recombinase activating gene-1 (RAG-1). Furthermore, the SP+ cell did express the message for the CD3 delta chain. Taken together, these data suggest that the SP+ cell line resulted from HTLV-I infection of a mature CD4+/CDB+ lymphocyte. This cell line can be potentially useful as a model, both for regulation of cellular functions by HTLV-I and for immunologic functions of mature dual CD4/CD8 positive T-cells.

Seroprevalence of human T lymphotropic virus type I in Puerto Rico.

Serum specimens from Puerto Rican residents were tested for antibodies to human T lymphotropic virus type I (HTLV-I) using an enzyme immunoassay, Western immunoblot, and radioimmunoprecipitation assays. Of 1,279 specimens obtained during a dengue virus surveillance program in 1986 and 1987, 3 (0.2%) tested positive; an additional 11 were indeterminate. Of 602 specimens obtained from blood donors in Ponce in 1987, 1 (0.2%) was positive; an additional specimen was indeterminate. Of 21 persons hospitalized for problems related to intravenous drug use in 1986 and 1987, 1 (5%) tested positive for HTLV-I antibodies.

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction.

The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models.