Identification of HTLV-I gag protease and its sequential processing of the gag gene product

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.     

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.     

Differential induction of the 200-family proteins in Daudi Burkitt's lymphoma cells by interferon-alpha

Interferon (IFN)-inducible "effector" proteins mediate the biological activities of the IFNs. Therefore, the identification of the functional role(s) of IFN inducible proteins in IFN action is important to elucidate the molecular mechanisms by which IFNs inhibit cell growth. One family (the "200-family") of IFN-inducible proteins includes structurally related murine (p202a, p202b, p203, p204 and D3) and human (MNDA, IFI-16 and AIM2) proteins. However, their role in IFN action remains to be established. Here we report that IFN-alpha treatment of Daudi Burkitt's lymphoma cells resulted in differential induction of MNDA, IFI 16, and a p202-related protein (p202RP). Interestingly, IFN induction of p202RP preceded the induction of MNDA and IFI 16 proteins and the growth inhibition by IFN. Additionally, the induction of these proteins by IFN was accompanied by: (i) a transient increase in p21(WAF1/CIP1) levels; (ii) an increase in the functional form of pRb and p130; (iii) an inhibition of the sequence-specific DNA binding activity of E2F complexes; and (iv) a marked decrease in c-Myc levels. Our observations reported herein provide support to the hypothesis that IFN-inducible p202RP and MNDA proteins from the 200-family contribute to the growth inhibitory activities of the IFNs.     

Aim2 deficiency stimulates the expression of IFN-inducible Ifi202, a lupus susceptibility murine gene within the Nba2 autoimmune susceptibility locus

Murine Aim2 and p202 proteins (encoded by the Aim2 and Ifi202 genes) are members of the IFN-inducible p200 protein family. Both proteins can sense dsDNA in the cytoplasm. However, upon sensing dsDNA, only the Aim2 protein through its pyrin domain can form an inflammasome to activate caspase-1 and induce cell death. Given that the p202 protein has been predicted to inhibit the activation of caspase-1 by the Aim2 protein and that increased levels of the p202 protein in female mice of certain strains are associated with lupus susceptibility, we compared the expression of Aim2 and Ifi202 genes between Aim2-deficient and age-matched wild-type mice. We found that the Aim2 deficiency in immune cells stimulated the expression of Ifi202 gene. The increased levels of the p202 protein in cells were associated with increases in the expression of IFN-β, STAT1, and IFN-inducible genes. Moreover, after knockdown of Aim2 expression in the murine macrophage cell line J774.A1, IFN-β treatment of cells robustly increased STAT1 protein levels (compared with those of control cells), increased the activating phosphorylation of STAT1 on Tyr-701, and stimulated the activity of an IFN-responsive reporter. Notably, the expression of Aim2 in non-lupus-prone (C57BL/6 and B6.Nba2-C) and lupus-prone (B6.Nba2-ABC) splenic cells and in a murine macrophage cell line that overexpressed p202 protein was found to be inversely correlated with Ifi202. Collectively, our observations demonstrate an inverse correlation between Aim2 and p202 expressions. We predict that defects in Aim2 expression within immune cells contribute to increased susceptibility to lupus.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.     

The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.     

RAPID SYNCYTIUM FORMATION BETWEEN HUMAN T-CELL LEUKAEMIA VIRUS TYPE-I (HTLV-I)-INFECTED T-CELLS AND HUMAN NERVOUS SYSTEM CELLS: A POSSIBLE IMPLICATION FOR TROPICAL SPASTIC PARAPARESIS/HTLV-I ASSOCIATED MYELOPATHY

Tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM), is characterized by infiltration of human T cell leukaemia virus type-I (HTLV-I)-infected T-cells, anti-HTLV-I cytotoxic T cells and macrophages into the patients’ cerebrospinal fluid and by intrathecally formed anti-HTLV-I antibodies. This implies that the disease involves a breakdown of the blood—brain barrier. Since astrocytes play a central role in establishing this barrier, the authors investigated the hypothesis that the HTLV-I infected T cells disrupt this barrier by damaging the astrocytes. The present study revealed the HTLV-I-producing T cells conferred a severe cytopatic effect upon monolayers of astrocytoma cell line in co-cultures. Following co-cultivation, HTLV-I DNA and proteins appeared in the monolayer cells, but after reaching a peak their level gradually declined. This appearance of the viral components was proved to result from a fusion of the astrocytic cells with the virus-producing T cells, whereas their subsequent decline reflected the destruction of the resulting syncytia. This fusion could be specifically blocked by anti HTLV-I Env antibodies, indicating that it was mediated by the viral Env proteins expressed on the surface of the virus-producing cells. Similar fusion was observed between the HTLV-I-producing cells and certain other human nervous system cell lines. If such fusion of HTLV-I-infected T cells occurs also with astrocytes and other nervous system cells in TSP/HAM patients, it may account, at least partially, for the blood—brain barrier breakdown and some of the neural lesions in this syndrome.     

Mutations introduced along the HTLV-I envelope gene result in a non-functional protein: a basis for envelope conservation?

The envelope protein of the human T-cell leukemia virus type I (HTLV-I) is highly conserved among the isolates sequenced so far, as opposed to what is observed for the human immunodeficiency virus (HIV) envelope. By linker insertion scanning, we have produced 33 random mutations along the HTLV-I envelope gene, cloned into a eukaryotic expression vector. The resulting envelope products were analysed by immunoprecipitation and syncytia formation after transfection into COS-1 cells. We show here that 25 out of 33 mutations result in a non-functional envelope product as assessed by the lack of ability to form syncytia. In the majority of these mutants, the processing of the envelope gp61 precursor into the mature gp45 and gp20 proteins was affected. We propose that conformational constraints for processing and fusion abilities tend to limit the variability of the HTLV-I envelope. In three mutants, processing was observed but no syncytia were formed. These mutations might affect regions important for HTLV-I envelope functions, such as the receptor binding region.     

Identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein p15E required for inhibition of lymphoproliferation and its similarity to gp21 of human T-lymphotropic virus types I and II.

Synthetic peptides containing portions of a highly conserved region of retroviral transmembrane proteins of human and animal retroviruses were tested for their ability to inhibit lymphoproliferation to determine the minimum amino acid sequence required. The previously reported immunosuppression mediated by the peptide CKS-17 was confirmed and further localized to a sequence of eight residues essentially identical to the sequence present in the transmembrane protein gp21 of human T-lymphotropic virus types I and II (HTLV-I and -II). To substantiate the physiological relevance of the inhibition of lymphoproliferation observed with the synthetic peptides and to relate this activity to the intact protein, we purified the Rauscher murine leukemia virus transmembrane protein p15E by immunoaffinity chromatography and report that this purified component presented in the form of protein micelles inhibited the interleukin-2-dependent proliferation of the murine T-cell line CTLL-2 in a dose-dependent manner, with a half-maximal inhibitory dose (ID50) of approximately 16 nM. In comparison, the ID50 concentration of a recombinant form of p15E required to inhibit lymphoproliferation was approximately 2.2 microM. The results reported here support the hypothesis that the transmembrane protein gp21 of HTLV-I and -II participates in the mechanism of immunosuppression previously reported for the transmembrane proteins of feline leukemia virus and other animal retroviruses. Thus, the transmembrane protein of HTLV-I, the etiological agent of adult T-cell leukemia-lymphoma, may be partially responsible for the immunocompromised clinical course of this disease that results in fatal opportunistic infections in a majority of cases.