Assessment of Bcl-xL, TAX, and HBZ Gene Expression in Adult T cell Leukemia/Lymphoma Patients.

Adult T cell leukemia/lymphoma is a malignancy with a poor prognosis caused by human T lymphocyte virus type 1 (HTLV-1) infection. Tax and HBZ are two major viral proteins that may be involved in oncogenesis by disrupting apoptosis. Because Bcl-xL plays an integral role in the anti-apoptotic pathway, this study examines the interaction between host apoptosis and oncoproteins. We investigated 37 HTLV-1-infected individuals, including 18 asymptomatic and 19 adult T cell leukemia/lymphoma (ATLL) subjects. mRNA was extracted and converted to cDNA from peripheral blood mononuclear cells, and then gene expression was determined using TaqMan q-PCR. Moreover, the HTLV-1 proviral load (PVL) was also measured using a commercial absolute quantification kit (Novin Gene, Iran). Data analysis revealed that the mean of TAX, HBZ, and PVL was significantly higher among the study groups (ATLL and carrier groups p = .003, p = .000, and p = .002 respectively). There was no statistical difference in Bcl-xL gene expression between the study groups (p = .323). It is proposed that this anti-apoptotic pathway may not be directly involved in the development of ATLL lymphoma. Bcl-xL, TAX, HBZ gene expression, and PVL can be utilized as prognostic markers.

Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

Human T-Cell Leukemia Virus Type 1 Oncogenesis between Active Expression and Latency: A Possible Source for the Development of Therapeutic Targets.

The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a type of cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through the body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5-10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM/TSP). ATL is characterized by a low viral expression and poor prognosis. The oncogenic mechanism triggered by HTLV-1 is extremely complex and the molecular pathways are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed Tcell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt regulated cell death, thereby exerting their transforming effects. This review provides updated information on the mechanisms underlying the transforming action of HTLV-1 and highlights potential therapeutic targets to combat ATL.

Mouse Models for HTLV-1 Infection and Adult T Cell Leukemia.

Adult T cell leukemia (ATL) is an aggressive hematologic disease caused by human T cell leukemia virus type 1 (HTLV-1) infection. Various animal models of HTLV-1 infection/ATL have been established to elucidate the pathogenesis of ATL and develop appropriate treatments. For analyses employing murine models, transgenic and immunodeficient mice are used because of the low infectivity of HTLV-1 in mice. Each mouse model has different characteristics that must be considered before use for different HTLV-1 research purposes. HTLV-1 Tax and HBZ transgenic mice spontaneously develop tumors, and the roles of both Tax and HBZ in cell transformation and tumor growth have been established. Severely immunodeficient mice were able to be engrafted with ATL cell lines and have been used in preclinical studies of candidate molecules for the treatment of ATL. HTLV-1-infected humanized mice with an established human immune system are a suitable model to characterize cells in the early stages of HTLV-1 infection. This review outlines the characteristics of mouse models of HTLV-1 infection/ATL and describes progress made in elucidating the pathogenesis of ATL and developing related therapies using these mice.

HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo.

HTLV-1 bZIP factor impairs DNA mismatch repair system.

Adult T-cell leukemia (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Microsatellite instability (MSI) has been observed in ATL cells. Although MSI results from impaired mismatch repair (MMR) pathway, no null mutations in the genes encoding MMR factors are detectable in ATL cells. Thus, it is unclear whether or not impairment of MMR causes the MSI in ATL cells. HTLV-1 bZIP factor (HBZ) protein interacts with numerous host transcription factors and significantly contributes to disease pathogenesis and progression. Here we investigated the effect of HBZ on MMR in normal cells. The ectopic expression of HBZ in MMR-proficient cells induced MSI, and also suppressed the expression of several MMR factors. We then hypothesized that the HBZ compromises MMR by interfering with a transcription factor, nuclear respiratory factor 1 (NRF-1), and identified the consensus NRF-1 binding site at the promoter of the gene encoding MutS homologue 2 (MSH2), an essential MMR factor. The luciferase reporter assay revealed that NRF-1 overexpression enhanced MSH2 promoter activity, while co-expression of HBZ reversed this enhancement. These results supported the idea that HBZ suppresses the transcription of MSH2 by inhibiting NRF-1. Our data demonstrate that HBZ causes impaired MMR, and may imply a novel oncogenesis driven by HTLV-1.

HBZ upregulates myoferlin expression to facilitate HTLV-1 infection.

The complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1), primarily infects CD4+ T-cells in vivo. Infectious spread within this cell population requires direct contact between virally-infected and target cells. The HTLV-1 accessory protein, HBZ, was recently shown to enhance HTLV-1 infection by activating intracellular adhesion molecule 1 (ICAM-1) expression, which promotes binding of infected cells to target cells and facilitates formation of a virological synapse. In this study we show that HBZ additionally enhances HTLV-1 infection by activating expression of myoferlin (MyoF), which functions in membrane fusion and repair and vesicle transport. Results from ChIP assays and quantitative reverse transcriptase PCR indicate that HBZ forms a complex with c-Jun or JunB at two enhancer sites within the MYOF gene and activates transcription through recruitment of the coactivator p300/CBP. In HTLV-1-infected T-cells, specific inhibition of MyoF using the drug, WJ460, or shRNA-mediated knockdown of MyoF reduced infection efficiency. This effect was associated with a decrease in cell adhesion and an intracellular reduction in the abundance of HTLV-1 envelope (Env) surface unit (SU) and transmembrane domain (TM). Lysosomal protease inhibitors partially restored SU levels in WJ460-treated cells, and SU localization to LAMP-2 sites was increased by MyoF knockdown, suggesting that MyoF restricts SU trafficking to lysosomes for degradation. Consistent with these effects, less SU was associated with cell-free virus particles. Together, these data suggest that MyoF contributes to HTLV-1 infection through modulation of Env trafficking and cell adhesion.

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes' pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Interplay between innate immunity and the viral oncoproteins Tax and HBZ in the pathogenesis and therapeutic response of HTLV-1 associated adult T cell leukemia.

The Human T-cell Leukemia virus type 1 (HTLV-1) causes an array of pathologies, the most aggressive of which is adult T-cell leukemia (ATL), a fatal blood malignancy with dismal prognosis. The progression of these diseases is partly ascribed to the failure of the immune system in controlling the spread of virally infected cells. HTLV-1 infected subjects, whether asymptomatic carriers or symptomatic patients are prone to opportunistic infections. An increasing body of literature emphasizes the interplay between HTLV-1, its associated pathologies, and the pivotal role of the host innate and adoptive immune system, in shaping the progression of HTLV-1 associated diseases and their response to therapy. In this review, we will describe the modalities adopted by the malignant ATL cells to subvert the host innate immune response with emphasis on the role of the two viral oncoproteins Tax and HBZ in this process. We will also provide a comprehensive overview on the function of innate immunity in the therapeutic response to chemotherapy, anti-viral or targeted therapies in the pre-clinical and clinical settings.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

Coevolution of HTLV-1-HBZ, Tax, and proviral load with host IRF-1 and CCNA-2 in HAM/TSP patients.

Background HTLV-1-associated myelopathy (HAM/TSP) is a progressive neurodegenerative inflammatory condition of HTLV-1 infection. Viral-host interactions are a significant contributor to the symptoms of HTLV-1-associated diseases. Therefore, in this study, the expression of the main regulatory viral factors and proviral load (PVL) and two host transcription molecules were evaluated in HAM/TSP patients. Materials and methods The study population included 17 HAM/TSP patients, 20 asymptomatic carriers (ACs), and 19 healthy controls (HCs). RNA and DNA were extracted from PBMCs for assessment of the gene expressions and PVL assessment using RT-qPCR and TaqMan method. Results HTLV-1-PVL was higher in HAM/TSPs (395.80 ± 99.69) than ACs (92.92 ± 29.41) (P = 0.001). The Tax expression in HAM/TSPs (7.8 ± 5.7) was strongly higher than ACs (0.06 ± 0.04) (P = 0.02), while HTLV-1-HBZ was only increased around three times in HAM/TSPs (3.17), compared to ACs (1.20) and not significant. The host IRF1 expression in HAM/TSPs (0.4 ± 0.31) was higher than ACs (0.09 ± 0.05) (P = 0.02) and also HCs (0.16 ± 0.07) (P = 0.5), but lower in ACs than HCs (p = 0.01). Although, in HAM/TSPs (0.13 ± 0.09) and ACs (0.03 ± 0.02) CCNA-2 expression was statistically fewer than HCs (0.18 ± 0.06) (P = 0.03, P = 0.001, respectively), in HAM/TSP was higher than ACs (P = 0.1), but did not meet a 95% confidence interval. Conclusion The study showed that HTLV-1-PVL and Tax, along with host IRF-1, could be considered biomarkers in HAM/TSP development. Furthermore, IRF-1, as an essential transcription factor, can be considered a pivotal target in HAM/TSPs treatment.

The Road to HTLV-1-Induced Leukemia by Following the Subcellular Localization of HTLV-1-Encoded HBZ Protein.

Human T cell leukemia virus-1 (HTLV-1) is the causative agent of a severe cancer of the lymphoid lineage that develops in 3-5% of infected individuals after many years. HTLV-1 infection may also induce a serious inflammatory pathology of the nervous system designated HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two virus-encoded proteins, the viral transactivator Tax-1 and the HTLV-1 basic leucine-zipper factor HBZ, are strongly involved in the oncogenic process. Tax-1 is involved in initial phases of the oncogenic process. Conversely, HBZ seems to be involved in maintenance of the neoplastic state as witnessed by the generation of leukemic/lymphomatous phenotype in HBZ transgenic mice and the persistent expression of HBZ in all phases of the oncogenic process. Nevertheless, the intimate molecular and cellular mechanism mediated by the two viral proteins, particularly HBZ, in oncogenesis still remain elusive. An important step toward the complete comprehension of HBZ-associated oncogenicity is the clarification of the anatomical correlates of HBZ during the various phases of HTLV-1 infection to development of HTLV-1-associated inflammatory pathology and ultimately to the establishment of leukemia. In this review, I will summarize recent studies that have established for the first time a temporal and unidirectional expression of HBZ, beginning with an exclusive cytoplasmic localization in infected asymptomatic individuals and in HAM/TSP patients and ending to a progressive cytoplasmic-to-nuclear transition in leukemic cells. These results are framed within the present knowledge of HTLV-1 infection and the future lines of research that may shed new light on the complex mechanism of HTLV-1- mediated oncogenesis.

Functional and Pathogenic Roles of Retroviral Antisense Transcripts.

Exogenous retroviruses such as human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia virus type 1 (HTLV-1) and bovine leukemia virus (BLV) can cause various diseases including immunodeficiency, inflammatory diseases and hematologic malignancies. These retroviruses persistently infect their hosts. Therefore, they need to evade host immune surveillance. One way in which these viruses might avoid immune detection is to utilize functional RNAs, rather than proteins, for certain activities, because RNAs are not recognized by the host immune system. HTLV-1 encodes the HTLV-1 bZIP factor (HBZ) gene in the antisense strand of the provirus. The HBZ protein is constantly expressed in HTLV-1 carriers and patients with adult T-cell leukemia-lymphoma, and it plays critical roles in pathogenesis. However, HBZ not only encodes this protein, but also functions as mRNA. Thus, HBZ gene mRNA is bifunctional. HIV-1 and BLV also encode long non-coding RNAs as antisense transcripts. In this review, we reshape our current understanding of how these antisense transcripts function and how they influence disease pathogenesis.

Regulation of HTLV-1 transformation.

Human T-cell leukemia virus type 1 (HTLV-1) is the only identified oncogenic human retrovirus. HTLV-1 infects approximately 5-10 million people worldwide and is the infectious cause of adult T-cell leukemia/lymphoma (ATL) and several chronic inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), dermatitis, and uveitis. Unlike other oncogenic retroviruses, HTLV-1 does not capture a cellular proto-oncogene or induce proviral insertional mutagenesis. HTLV-1 is a trans-activating retrovirus and encodes accessory proteins that induce cellular transformation over an extended period of time, upwards of several years to decades. Inarguably the most important viral accessory protein involved in transformation is Tax. Tax is a multifunctional protein that regulates several different pathways and cellular processes. This single viral protein is able to modulate viral gene expression, activate NF-κB signaling pathways, deregulate the cell cycle, disrupt apoptosis, and induce genomic instability. The summation of these processes results in cellular transformation and virus-mediated oncogenesis. Interestingly, HTLV-1 also encodes a protein called Hbz from the antisense strand of the proviral genome that counters many Tax functions in the infected cell, such as Tax-mediated viral transcription and NF-κB activation. However, Hbz also promotes cellular proliferation, inhibits apoptosis, and disrupts genomic integrity. In addition to viral proteins, there are other cellular factors such as MEF-2, superoxide-generating NAPDH oxidase 5-α (Nox5α), and PDLIM2 which have been shown to be critical for HTLV-1-mediated T-cell transformation. This review will highlight the important viral and cellular factors involved in HTLV-1 transformation and the available in vitro and in vivo tools used to study this complex process.

Stephen Oroszlan and Retroviral Proteins.

Stephen Oroszlan received his early education in Hungary, graduating in 1950 from the Technical University in Budapest with a degree in chemical engineering [...].

Does HTLV-1 Infection Show Phenotypes Found in Sjögren's Syndrome?

Viruses are a possible cause for Sjögren's syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.

Immune suppression of vaccine-induced CD8+ T-cell responses by gamma retrovirus envelope is mediated by interleukin-10-producing CD4+ T cells.

Retroviral envelope (Env) proteins have long been recognized to exhibit immunosuppressive properties, which affect the CD8+ T-cell response to an infection but also to immunization. Interestingly, we previously showed in the Friend murine leukemia virus (F-MuLV) model that the surface Env protein gp70 also plays a role in immunosuppression, in addition to the immunosuppressive function attributed to the transmembrane Env protein. We now demonstrate that immunization with F-MuLV Env leads to a significant increase in interleukin-10 (IL-10)-producing CD4+ T cells and that the induction of CD8+ T-cell responses in the presence of Env is rescued if the capacity of CD4+ T cells to produce IL-10 is abrogated, indicating a mechanistic role of IL-10-producing CD4+ T cells in mediating the Env-induced suppression of CD8+ T-cell responses in Env co-immunization. We found that CD8+ T-cell responses against different immunogens are not all equally affected. On the other hand, suppression of immunity was observed not only in co-immunization experiments but also for immune control of subcutaneous tumor growth after an Env immunization. Finally, we show that suppression of CD8+ T cells by the surface Env protein is observed not only for Friend MuLV Env but also for the Env proteins of other gamma retroviruses. Taken together, our results show that IL-10-producing CD4+ T cells mechanistically underlie the Env-mediated suppression of CD8+ T-cell responses and suggest the presence of an immunosuppressive motif in the surface Env protein of gamma retroviruses.

Stephan Oroszlan and the Proteolytic Processing of Retroviral Proteins: Following A Pro.

Steve Oroszlan determined the sequences at the ends of virion proteins for a number of different retroviruses. This work led to the insight that the amino-terminal amino acid of the mature viral CA protein is always proline. In this remembrance, we review Steve's work that led to this insight and show how that insight was a necessary precursor to the work we have done in the subsequent years exploring the cleavage rate determinants of viral protease processing sites and the multiple roles the amino-terminal proline of CA plays after protease cleavage liberates it from its position in a protease processing site.

Retroviral Antisense Transcripts and Genes: 33 Years after First Predicted, a Silent Retroviral Revolution?

Paradigm shifts throughout the history of microbiology have typically been ignored, or met with skepticism and resistance, by the scientific community. This has been especially true in the field of virology, where the discovery of a "contagium vivum fluidum", or infectious fluid remaining after excluding bacteria by filtration, was initially ignored because it did not coincide with the established view of microorganisms. Subsequent studies on such infectious agents, eventually termed "viruses", were met with skepticism. However, after an abundance of proof accumulated, viruses were eventually acknowledged as defined microbiological entities. Next, the proposed role of viruses in oncogenesis in animals was disputed, as was the unique mechanism of genome replication by reverse transcription of RNA by the retroviruses. This same pattern of skepticism holds true for the prediction of the existence of retroviral "antisense" transcripts and genes. From the time of their discovery, it was thought that retroviruses encoded proteins on only one strand of proviral DNA. However, in 1988, it was predicted that human immunodeficiency virus type 1 (HIV-1), and other retroviruses, express an antisense protein encoded on the DNA strand opposite that encoding the known viral proteins. Confirmation came quickly with the characterization of the antisense protein, HBZ, of the human T-cell leukemia virus type 1 (HTLV-1), and the finding that both the protein and its antisense mRNA transcript play key roles in viral replication and pathogenesis. However, acceptance of the existence, and potential importance, of a corresponding antisense transcript and protein (ASP) in HIV-1 infection and pathogenesis has lagged, despite gradually accumulating theoretical and experimental evidence. The most striking theoretical evidence is the finding that asp is highly conserved in group M viruses and correlates exclusively with subtypes, or clades, responsible for the AIDS pandemic. This review outlines the history of the major shifts in thought pertaining to the nature and characteristics of viruses, and in particular retroviruses, and details the development of the hypothesis that retroviral antisense transcripts and genes exist. We conclude that there is a need to accelerate studies on ASP, and its transcript(s), with the view that both may be important, and overlooked, targets in anti-HIV therapeutic and vaccine strategies.