Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes.

Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression.     

Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses.

Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses.     

A chimeric human T-cell lymphotropic virus type 1 with the envelope glycoprotein of Moloney murine leukemia virus is infectious for murine cells

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.     

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry

Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.     

Evolutionary strategies of human T-cell lymphotropic virus type II

Human T-cell lymphotropic virus type II (HTLV-II) primarily infects two different populations in which the virus is transmitted in very diverse ways. In endemically infected populations, the virus is propagated through sexual contact, and by mother to child transmission via breast-feeding, among intravenous drug users (IDUs), spread is mainly due to blood-borne transmission via needle sharing. The phylogeny of HTLV-II strains isolated from American Indian and Pygmy tribes and strains from IDUs, reveal that the virus originated on the African continent as a result of a simian to human transmission at least 400,000 years ago. HTLV-II was very likely introduced into the American continent during one or more migrations of HTLV-II infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. During the last few decades, HTLV-II has been transmitted from native American Indians to IDUs at least twice, followed by a rapid spread of the virus in the drug users population world-wide due to the practice of needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates, with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7x10(-4) compared to 1.7/7.3x10(-7) nucleotide substitutions per site per year in the LTR region. Although many of the HTLV-II infected drug users are co-infected with HIV, the dramatic acceleration of the evolutionary rate seems to be mainly related to the different modes of transmission in the two populations. These contrasting evolutionary rates correlate with an endemic spread of HTLV-II in infected tribes compared to an epidemic spread in IDUs.     

The role of cytotoxic T lymphocytes in human T-cell lymphotropic virus type 1 infection

The vast majority of individuals infected with human T-cell lymphotropic virus type 1 have a strong and persistently activated cytotoxic T lymphocyte response to the virus. Experimental work investigating the effects of human T-cell lymphotropic virus-specific cytotoxic T lymphocytes is conflicting. One significant body of work suggests that specific cytotoxic T lymphocytes are protective and help to reduce the risk of disease. However, another body of work implies that specific cytotoxic T lymphocytes play an important role in the development of disease. Here we use a theoretical model to explore the role of cytotoxic T lymphocytes in persistent infection. A way of reconciling the apparently contradictory data is proposed and experimentally testable predictions are made.     

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.     

A 10-amino acid domain within human T-cell leukemia virus type 1 and type 2 tax protein sequences is responsible for their divergent subcellular distribution

Human T-cell leukemia virus type 1 and type 2 (HTLV-1/2) are related retroviruses that infect T-lymphocytes. Whereas HTLV-1 infection can cause leukemia, HTLV-2 has not been demonstrated to be the agent of a hematological malignant disease. Nevertheless, the virally encoded Tax-1 and Tax-2 transactivators display a high percentage of similarity. Tax-1 is a shuttling protein that contains a noncanonical nuclear localization signal as well as a nuclear export signal. The presence of the nuclear localization signal and the nuclear export signal domains in the Tax-2 sequence has not been determined. The distribution of Tax-2 in infected cells is not known but has been assumed to be similar to that of Tax-1. By using a Tax-2-specific antibody, we report here that Tax-2 is located predominantly in the cytoplasm of the HTLV-2 immortalized or transformed infected T-cells. These results were confirmed after transient transfection of untagged Tax-1 and Tax-2 constructs, histidine tag Tax1/Tax2, GFP-Tax, and Tax-GFP fusion constructs in several cell lines. We show that this unanticipated localization is not due to a default in the Tax-2 nuclear localization signal functions nor to major differences in Tax-2 versus Tax-1 binding to the IKKgamma/NEMO protein. In addition, we demonstrate that inhibiting the proteasome results in a relocalization of Tax-1 in the cytoplasm, similar to that of Tax-2. By using a series of Tax-1/Tax-2 chimeras, we determined that the minimal domain that is necessary for Tax-2 peculiar distribution encompasses amino acids 90-100. Finally, we show a high correlation between intracellular localization of Tax and their NF-kappaB or CREB transactivating ability.     

Hepatitis C virus core protein interacts with cellular putative RNA helicase

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.     

The simian origins of the pathogenic human T-cell lymphotropic virus type I

At least four, and possibly six, molecular subtypes of human T-cell lymphotropic virus type I (HTLV-I) exist: one is confined to Melanesia/Australia, one is ubiquitous, and the others are found only in Africa. Molecular epidemiology suggests that all subtypes arose from separate interspecies transmissions from simians to humans.     

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.     

Human T-cell lymphotropic virus type I and adult T-cell leukemia/lymphoma outside Japan and the Caribbean Basin

Ninety-six patients with the diagnosis of adult T-cell leukemia/lymphoma (ATLL) were identified in countries outside Japan and the Caribbean Basin. Seventy-four of these patients were initially diagnosed in the United States; 25 of 52 patients whose places of birth were known had been born in the United States. The detection of 14 patients born in the southeastern United States, all black, indicates a group deserving particular attention for studies of human T-cell lymphotropic virus type I (HTLV-I), a suspected etiologic agent in most cases of ATLL. Although geographic clustering of ATLL in areas endemic for HTLV-I, particularly southwest Japan and the Caribbean Basin, is a dramatic feature of this disease, a review of the literature indicates that HTLV-I-associated ATLL probably occurs sporadically in a much wider distribution, the disease being diagnosed in native-born African, Chinese, European, and Latin American patients. A registry for ATLL cases is suggested, to assist in the identification of risk factors for this disease and, at the same time, improve case definitions and early diagnosis.     

In vivo cellular tropism of human T-cell leukemia virus type 1.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.