Alix de Brogniez

Preventive and Therapeutic Strategies for Bovine Leukemia Virus: Lessons for HTLV

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads.

Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.

Vaccination against δ−Retroviruses: The Bovine Leukemia Virus Paradigm

Bovine leukemia virus (BLV) and human T-lymphotropic virus type 1 (HTLV-1) are closely related δ-retroviruses that induce hematological diseases. HTLV-1 infects about 15 million people worldwide, mainly in subtropical areas. HTLV-1 induces a wide spectrum of diseases (e.g., HTLV-associated myelopathy/tropical spastic paraparesis) and leukemia/lymphoma (adult T-cell leukemia). Bovine leukemia virus is a major pathogen of cattle, causing important economic losses due to a reduction in production, export limitations and lymphoma-associated death. In the absence of satisfactory treatment for these diseases and besides the prevention of transmission, the best option to reduce the prevalence of δ-retroviruses is vaccination. Here, we provide an overview of the different vaccination strategies in the BLV model and outline key parameters required for vaccine efficacy.

Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo

Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host.

Recent advances in BLV research

Different animal models have been proposed to investigate the mechanisms of Human T-lymphotropic Virus (HTLV)-induced pathogenesis: rats, transgenic and NOD-SCID/γcnull (NOG) mice, rabbits, squirrel monkeys, baboons and macaques. These systems indeed provide useful information but have intrinsic limitations such as lack of disease relevance, species specificity or inadequate immune response. Another strategy based on a comparative virology approach is to characterize a related pathogen and to speculate on possible shared mechanisms. In this perspective, bovine leukemia virus (BLV), another member of the deltaretrovirus genus, is evolutionary related to HTLV-1. BLV induces lymphoproliferative disorders in ruminants providing useful information on the mechanisms of viral persistence, genetic determinants of pathogenesis and potential novel therapies.

Plant polysaccharides initiate underground crosstalk with bacilli by inducing synthesis of the immunogenic lipopeptide surfactin.

Some plant-associated bacteria such as Bacillus sp. can protect their host from pathogen ingress and this biocontrol activity correlates with their potential to form multiple antibiotics upon in vitro growth. However, our knowledge on antibiotic production by soil bacilli evolving on roots in natural conditions is still limited. In this work, antibiome imaging first revealed that the lipopeptide surfactin is the main bacterial ingredient produced in planta within the first hours of interaction with root tissues. We further demonstrated that surfactin synthesis is specifically stimulated upon perception of plant cell wall polymers such as xylan or arabinogalactan, leading to fast accumulation of micromolar amounts in the root environment. At such concentrations, the lipopeptide may not only favour the ecological fitness of the producing strain in term of root colonization, but also triggers systemic resistance in the host plant. This surfactin-induced immunity primes the plant to better resist further pathogen ingress, and involves only limited expression of defence-related molecular events and does not provoke seedling growth inhibition. By contrast with the strong response mounted upon perception of pathogens, this strongly attenuated defensive reaction induced by surfactin in plant tissues should help Bacillus to be tolerated as saprophytic partner by its host.

Mutation of a Single Envelope N-Linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus

ABSTRACT Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans.

Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis

Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis.

The bovine leukemia virus microRNAs permit escape from innate immune response and contribute to viral replication in the natural host

In the natural host (Bos taurus), infection with bovine leukemia virus (BLV) remains mostly asymptomatic, resulting in a benign lymphocytosis only in about one-third of infected animals and even less frequently in a B-cell leukemia/lymphoma (10% of cases). BLV can also be experimentally transmitted to sheep that almost invariably develop leukemia/lymphoma after shorter latency periods. Upon integration, the BLV provirus becomes transcriptionally silent except for the microRNAs that remain very abundantly expressed. We used reverse genetics to evaluate the role of the viral microRNAs in the natural and experimental hosts. A BLV lacking the microRNAs replicated at wild-type levels in sheep, indicating that these sequences were dispensable. Surprisingly, the microRNAs were required for efficient replication in cows, thereby underlining the importance of studying viral determinants in the natural host. To understand the mechanisms associated with the microRNAs, we performed high-throughput RNA-sequencing of transgenic B cell lines and peripheral blood mononuclear cells isolated from cows infected either by wild-type or by isogenic microRNA-deleted viruses. Bioinformatic analyses revealed that BLV microRNAs modulate a series of pathways that include B-cell signaling and immunity. Reporter assays showed that the microRNAs target granzyme A and c-FOS transcripts and downregulate indirectly Annexin A1 and phosphoinositide-3-kinase PIK3CG. Finally, expression of the microRNAs in B-lymphocytes was associated with a decrease in phagocytosis by primary bovine macrophages. These studies thus assign a functional relevance of the BLV microRNAs in viral evasion from the innate immune response in its natural host.