High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1.

Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leukemia but is also responsible for severe neurodegenerative and endocrine disorders. Combatting HTLV-1 infections requires a detailed understanding of the viral mechanisms in the host. Therefore, in vitro studies of important virus-encoded proteins would be critical. Our focus herein is on the HTLV-1-encoded regulatory protein p13II, which interacts with the inner mitochondrial membrane, increasing its permeability to cations (predominantly potassium, K+). Thereby, this protein affects mitochondrial homeostasis. We report on our progress in developing specific protocols for heterologous expression of p13II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13II C-terminus was instrumental in increasing the persistently low expression of soluble p13II in its native form. We subsequently developed approaches for protein spin labeling and a conformation study using double electron-electron resonance (DEER) spectroscopy and a fluorescence-based cation uptake assay for p13II in liposomes. Our DEER results point to large protein conformation changes occurring upon transition from the soluble to the membrane-bound state. The functional assay on p13II-assisted transport of thallium (Tl+) through the membrane, wherein Tl+ substituted for K+, suggests transmembrane potential involvement in p13II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13II and would aid in the development of structure-based protein inhibitors and markers.

Expression of HTLV-1 Genes in T-Cells Using RNA Electroporation.

Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

Evaluation of HTLV-1 HBZ and proviral load, together with host IFN λ3, in pathogenesis of HAM/TSP.

Human T-cell lymphotropic virus 1 (HTLV-1) is associated with two progressive diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Although HTLV-1 proviral load (PVL) has been introduced as a risk factor for these diseases' progression, it is not sufficient on its own to yield an accurate estimation of the outcome of the infection. In the present study, PVL and HTLV-1 basic leucine zipper factor (HBZ) expression level as viral factors, and IFN λ3 as a host factor, were evaluated in HAM/TSP patients and HTLV-1 asymptomatic carriers (ACs). During 2014-2015, 12 HAM/TSP patients and 18 ACs who had been referred to the HTLV-1 Clinic, Ghaem Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran, were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and the DNA and mRNA were extracted for quantification of HBZ, IFN λ3 expression, and PVL using real-time PCR (TaqMan method). Although the PVL was higher in the HAM/TSP group, with a 94% confidence interval, there were no considerable differences in terms of HBZ mRNA and PVL between ACs and HAM patients. IFN λ3 expression in the HAM/TSP group was significantly higher than in the ACs (P = 0.02). To the best of our knowledge, no study has evaluated the expression level of IFN λ3 in HTLV-1 positive patients. The immune response against HTLV-1 viral antigens and virulent factors will therefore further refine our knowledge of interactions between the virus and host in the pathogenesis of HTLV-1-related disorders. The virus PVL and the host IFN λ3 can be used as pathogenic factors of HTLV-1 infected patients at risk of HAM/TSP manifestation. J. Med. Virol. 89:1102-1107, 2017. © 2016 Wiley Periodicals, Inc.

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

The HTLV-1 hbz antisense gene indirectly promotes tax expression via down-regulation of p30(II) mRNA.

Human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ) is transcribed from the antisense genomic DNA strand and functions differently in its RNA and protein forms. To distinguish between the roles of hbz mRNA and HBZ protein, we generated mutants in a proviral clone that specifically disrupt the hbz gene product. A proviral clone with a splice acceptor mutation that disrupts expression of the predominant hbz mRNA resulted in lower levels of tax mRNA. Heterologous hbz expression restored Tax activity in cells expressing this mutant clone. In contrast, proviral mutants that disrupt HBZ protein did not affect levels of tax mRNA. Expression of hbz resulted in lower levels of p30(II) mRNA. Mutation of p30(II) overcame the effects of the splice acceptor mutation of hbz, and restored tax expression. Thus, there is a complex interplay of viral regulatory proteins controlling levels of HTLV-1 gene expression.

Direct comparison of antigen production and induction of apoptosis by canarypox virus- and modified vaccinia virus ankara-human immunodeficiency virus vaccine vectors.

Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.

Inter-clade cross-reactivity of HIV-1-specific T cell responses in human immunodeficiency virus type 1 infection in China.

To determine the degree of HIV-1-specific cytotoxic-T-lymphocyte (CTL) cross-responses to the clade B and C consensus sequences at the single peptide level. We assessed CTL responses in 46 HIV-1 clade B chronically infected individuals using an interferon-gamma Elispot assay with a total of 826 overlapping peptides spanning HIV-1 clade B and C consensus sequences. In general, 583 peptides were recognized by HIV-1-specific T cells in the study subjects (292 clade B, 291 clade C respectively), of which 204 peptides in both clades were recognized simultaneously. The HIV-1-specific CTL responses to both clade peptides contributed 54.23% (954/1759) to the total responses. No significant difference was observed between the overall magnitude or frequency of CTL responses to clade B proteins and those to clade C proteins. According to the profiles of CTL magnitude and CTL frequency, the top 44 and 35 synthetic peptides were identified as immunodominant regions in the clade B and C consensus sequences respectively and 27 corresponding peptides in two immunodominant regions were cross-reactive. These peptides with cross-reactivity had a significantly higher ability to elicit CTL responses (P< 0.01) and preferentially had a trend of lower entropy and higher inter-clade homology. A wide degree of cross-clade reactivity of HIV-1-specific T cells exist in clade B and clade C variants. Most of immunodominant peptides with cross-reactivity are vigorous to elicit CTL responses and preferentially be conservative. This result may make future HIV-1 vaccines including multiple copies of CTL epitopes in these immunodominant peptides effective for this population.

Host factors exploited by retroviruses.

Retroviruses make a long and complex journey from outside the cell to the nucleus in the early stages of infection, and then an equally long journey back out again in the late stages of infection. Ongoing efforts are identifying an enormous array of cellular proteins that are used by the viruses in the course of their travels. These host factors are potential new targets for therapeutic intervention.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products.

BACKGROUND: While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). RESULTS: Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. CONCLUSION: HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development.

An internal ribosome entry site promotes translation of a novel SIV Pr55(Gag) isoform.

In complex retroviruses including simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1), the major structural proteins are encoded by the gag gene and translated as a precursor polyprotein, Pr55(Gag). An internal ribosome entry site (IRES) within the coding region of HIV-1 and HIV type 2 (HIV-2) gag RNA mediates expression of N-terminally truncated isoforms of the precursor polyprotein. In this study, we identify an N-terminally truncated SIV Pr55(Gag) isoform expressed from the SIV gag gene SIV p43. We demonstrate that translation of p43 occurs independently of Pr55(Gag) translation and initiates at an in-frame AUG within the gag transcript. We test several mechanisms that could mediate translation of p43 and report that translation of SIV p43 is driven by an IRES located entirely within the coding region of gag mRNA. Additionally, we present data that suggest SIV p43 affects viral replication in cell culture.

[Molecular cloning of a novel retroviral NP9 gene of human endogenous retrovirus and its viral protein expression].

OBJECTIVE: To investigate the relationship between retroviruses and autoimmune diseases, to clone the novel retroviral NP9 gene from human endogenous retrovirus (HERV), and to construct its expression vector. METHODS: The viral NP9 gene was amplified and cloned by RT-PCR and T-A clone techniques, and its sequence was determined with Perkin-Elmer 377 DNA Sequencer. The amplified viral NP9 gene was subcloned into the prokaryotic express vector pQE30. The recombinant plasmids were identified by restriction endonuclease digestion and sequencing. The recombinant pQE30-NP9 protein was expressed in M15 host cells under the IPTG induction and showed with SDS-PAGE,and the corresponding NP9 viral protein was identified with Western blot analysis. RESULT: A specific band of 250 bp was amplified using RT-PCR from total RNA of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) and confirmed as the NP9 gene via T-A clone and DNA sequencing analyses. SDS-PAGE profile showed a clear protein band with a relative molecular weight 9 kD in the IPTG-induced samples, which was confirmed as viral NP9 protein by Western blot analysis. CONCLUSION: The NP9 gene has been successfully isolated and cloned from PBMCs of SLE patients and the corresponding NP9 viral protein expressed in prokaryotic expression vector.

Effect of 17beta-estradiol and progesterone on the expression of FeLV in chronically infected cells.

In a previous study, it was found that even though more male cats were infected by feline leukaemia virus (FeLV), females seemed to progress easier to overt disease. To study the effect of female hormones, 17beta-estradiol and progesterone were added in different concentrations (10(-3) M to 10(-12) M) to a culture of persistently FeLV-infected cells. The effect of both hormones was very similar. After 24 h the cell viability was very low at 10(-3) M and 10(-4) M but similar to controls at the remaining concentrations. Liberation of viral particles was estimated by the reverse transcriptase activity (RT), which was the lowest also at 10(-3) M and 10(-4) M. However, low viability could not account for this low RT, as when cells were lysed with lysis buffer RT was high. Thus, cells were dying without freeing viral particles, suggestive of apoptosis. This possibility was confirmed by staining hormone-treated cells with annexin V and propidium iodide. The FeLV antigen p27 measured in the cultures had a maximum at 10(-3) M and 10(-4) M, higher than controls and lysed cells, so the presence of p27 in the supernatant was not only due to cell lysis but a consequence of hormone effect. In conclusion, 17beta-estradiol and progesterone induce death of FeLV-infected cells at high concentrations, probably through a process of apoptosis, which might limit the spread of the infection, as infective viral particles would be hampered from budding.

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.

Evidence for the chronic in vivo production of human T cell leukemia virus type I Rof and Tof proteins from cytotoxic T lymphocytes directed against viral peptides.

Human T cell leukemia virus type I (HTLV-I) is a persistent virus that causes adult T cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Studies on rabbits have shown that viral proteins encoded by the open reading frames pX-I and pX-II are required for the establishment of the persistent infection. To examine the in vivo production of these proteins in humans, we have investigated whether cytotoxic T lymphocytes isolated from HTLV-I-infected individuals recognized pX-I and pX-II peptides. CD8(+) T lymphocytes to pX-I and pX-II peptides were detected in HTLV-I-infected individuals, whatever their clinical status, and even in the absence of any antigenic restimulation. These findings indicate that the HTLV-I pX-I and pX-II proteins are chronically synthesized in vivo, and are targets of the natural immune response to the virus.

High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis.

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.

Gene transfer using replication-defective human foamy virus vectors.

Replication-defective vectors based on an infectious molecular clone of human foamy virus (HFV) were constructed by deletion and replacement of the accessory genes with expression cassettes for puromycin-resistance and beta-glucouronidase. Cell lines which produced in excess of 10(5) helper virus-free transducing units/ml were generated by trans-complementation of the replication defect using a BHK-21-derived cell line expressing the Bel-1 transactivator. Vectors based on the HFV genome may provide useful alternatives to existing retroviral vectors.

Involvement of a spliced and defective human foamy virus in the establishment of chronic infection.

Human foamy retrovirus (HFV) is found as two proviruses (HFV and delta HFV) which differ by a splice-induced deletion within the bel1 transactivator gene. The defective delta HFV (which lacks a functional Bel1 but harbors an intronless bet gene) is predominantly found in nonlytic infections in vitro as well as in vivo. Here, we show that infection of cell lines stably transduced by delta HFV DNA with the highly lytic HFV leads to chronic infections characterized by an absence of lysis, a balanced ratio of HFV to delta HFV, and a persistent Bet expression accompanied by a shutoff of structural genes. While this system only partially reflects the natural situation, in which target cells are infected by HFV and delta HFV simultaneously, it strongly suggests that delta HFV is a defective interfering retrovirus. Accordingly, previous or concomitant exposure to delta HFV viruses greatly enhances the formation of lysis-resistant clones in culture after HFV infection. The inability of delta HFV proviruses encoding a mutated bet gene to induce chronic infection suggests a role for Bet in this process. Through a specific, splice-induced, genomic deletion, resulting in a switch from Bel1 to Bet expression, the lytic properties of HFV are progressively lost. Such programmed inactivation of a key gene represents a new regulatory mechanism of gene expression in retroviruses.

Abundance of an endogenous retroviral envelope protein in placental trophoblasts suggests a biological function.

To investigate the hypothesis that the human endogenous sequence ERV-3 has a function, we have cloned and expressed the transmembrane region of its envelope gene and raised specific antibodies to the fusion protein and to a synthetic peptide. These antibodies reacted with a 65-kDa polypeptide which constituted approximately 0.1% of the cellular protein in syncytiotrophoblasts in placenta. The evolutionary conservation and abundant expression of this endogenous retroviral protein in a specific cell type support the concept of a biological function. The similarity of a domain of ERV-3 env to putative immunosuppressive p15E sequences suggests that ERV-3 might form part of the placental immunosuppressive barrier between mother and foetus.

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.