Inhibition Of Viral Gene Expression Research Articles

Stabilization of RNA G-quadruplexes in the SARS-CoV-2 genome inhibits viral infection via translational suppression.

The G-quadruplex (G4) formed in single-stranded DNAs or RNAs plays a key role in diverse biological processes and is considered as a potential antiviral target. In the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 25 putative G4-forming sequences are predicted; however, the effects of G4-binding ligands on SARS-CoV-2 replication have not been studied in the context of viral infection. In this study, we investigated whether G4-ligands suppressed SARS-CoV-2 replication and whether their antiviral activity involved stabilization of viral RNA G4s and suppression of viral gene expression. We found that pyridostatin (PDS) suppressed viral gene expression and genome replication as effectively as the RNA polymerase inhibitor remdesivir. Biophysical analyses revealed that the 25 predicted G4s in the SARS-CoV-2 genome formed a parallel G4 structure. In particular, G4-644 and G4-3467 located in the 5' region of ORF1a, formed a G4 structure that could be effectively stabilized by PDS. We also showed that PDS significantly suppressed translation of the reporter genes containing these G4s. Taken together, our results demonstrate that stabilization of RNA G4s by PDS in the SARS-CoV-2 genome inhibits viral infection via translational suppression, highlighting the therapeutic potential of G4-ligands in SARS-CoV-2 infection.

The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation.

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription.

Intrinsic antiviral host factors confer cellular defense by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses ICP0, which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection, but mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type (WT) virus and mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5 which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors, and reveal that viral countermeasure can overcome SLFN antiviral activity.

Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing

Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated “base editors” (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.

Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity.

The HIRA histone chaperone complex deposits histone H3.3 into nucleosomes in a DNA replication- and sequence-independent manner. As herpesvirus genomes enter the nucleus as naked DNA, we asked whether the HIRA chaperone complex affects herpesvirus infection. After infection of primary cells with HSV or CMV, or transient transfection with naked plasmid DNA, HIRA re-localizes to PML bodies, sites of cellular anti-viral activity. HIRA co-localizes with viral genomes, binds to incoming viral and plasmid DNAs and deposits histone H3.3 onto these. Anti-viral interferons (IFN) specifically induce HIRA/PML co-localization at PML nuclear bodies and HIRA recruitment to IFN target genes, although HIRA is not required for IFN-inducible expression of these genes. HIRA is, however, required for suppression of viral gene expression, virus replication and lytic infection and restricts murine CMV replication in vivo. We propose that the HIRA chaperone complex represses incoming naked viral DNAs through chromatinization as part of intrinsic cellular immunity.

The variances of Sp1 and NF-κB elements correlate with the greater capacity of Chinese HIV-1 B'-LTR for driving gene expression.

The 5′ end of HIV-1 long terminal repeat (LTR) serves as a promoter that plays an essential role in driving viral gene transcription. Manipulation of HIV-1 LTR provides a potential therapeutic strategy for suppressing viral gene expression or excising integrated provirus. Subtype-specific genetic diversity in the LTR region has been observed. The minor variance of LTR, particularly in the transcription factor binding sites, can have a profound impact on its activity. However, the LTR profiles from major endemic Chinese subtypes are not well characterized. Here, by characterizing the sequences and functions of LTRs from endemic Chinese HIV-1 subtypes, we showed that nucleotide variances of Sp1 core promoter and NF-κB element are associated with varied LTR capacity for driving viral gene transcription. The greater responsiveness of Chinese HIV-1 B′-LTR for driving viral gene transcription upon stimulation is associated with an increased level of viral reactivation. Moreover, we demonstrated that the introduction of CRISPR/dead Cas9 targeting Sp1 or NF-κB element suppressed viral gene expression. Taken together, our study characterized LTRs from endemic HIV-1 subtypes in China and suggests a potential target for the suppression of viral gene expression and a novel strategy that facilitates the accomplishment of a functional cure.

Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge

Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.

An Ounce of Tat Prevention Is Worth a Pound of Functional Cure.

Infection by HIV-1 can effectively be suppressed by antiretroviral therapy (ART). However, the persistence of a small, latent virus reservoir impedes viral eradication. Thus, most infected individuals must be treated with ART for life. While efforts to find a sterilizing cure that eradicates the virus are ongoing, an alternative approach deemed a functional cure has remained less explored. A functional cure for HIV is envisioned as long-term control of virus replication in the absence of ART (reviewed in references 1 and 2). Such a functional cure might encompass effective immune interventions, reduction of the reservoir to a highly “controllable” size, and/or induction of a long-lasting suppression of viral gene expression. The Tat viral protein is a strong transactivator of viral gene expression, and viral replication is severely restricted in its absence; thus, it makes a logical antiviral target. A recent article by Mousseau et al. (3) reports on a novel Tat inhibitor that the authors show has the unusual ability to induce a “permanent state of latency.” Didehydro-cortistatin A (dCA) is a departure from current FDA-approved antiretroviral drugs in that it inhibits viral gene expression from integrated viruses. For the above reasons, dCA represents a novel inhibitor class. Cortistatin A (CA) is a steroidal alkaloid from Corticium simplex , a marine sponge, which has raised interest based on its antiproliferative effects (4). dCA is a derivative of CA that was reported to be equipotent (5) to the parent compound as well as relatively easy to obtain by chemical synthesis. One of the salient features of CA is its ability to bind to and inhibit cyclin-dependent kinase 11 (CDK11) with nanomolar affinity (6). CDK11 was recently shown by Valente and colleagues to be essential for HIV-1 gene expression by phosphorylating factors required for proper 3′ processing of viral mRNAs (7). Based …

CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.

Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs).

Production of cytokines plays an important role in the immune response.Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha.Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to detect an antigen specific response, making it an extremely flexible and versatile method. This capability, combined with the high throughput nature of the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The principle steps of intracellular cytokine staining is as follows: Cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail; An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell; Next, EDTA is added to remove adherent cells from the activation vessel;After washing, antibodies to cell surface markers can be added to the cells;The cells are then fixed in paraformaldehyde and permeabilized;The anti-cytokine antibody is added and the cells can be analyzed by flow cytometer.

Pyrrolidine Dithiocarbamate Inhibits Herpes Simplex Virus 1 and 2 Replication, and Its Activity May Be Mediated through Dysregulation of the Ubiquitin-Proteasome System

Pyrrolidine dithiocarbamate (PDTC) is widely used as an antioxidant or an NF-κB inhibitor. It has been reported to inhibit the replication of human rhinoviruses, poliovirus, coxsackievirus, and influenza virus. In this paper, we report that PDTC could inhibit the replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2). PDTC suppressed the expression of HSV-1 and HSV-2 viral immediate early (IE) and late (membrane protein gD) genes and the production of viral progeny. This antiviral property was mediated by the dithiocarbamate moiety of PDTC and required the presence of Zn(2+). Although PDTC could potently block reactive oxygen species (ROS) generation, it was found that this property did not contribute to its anti-HSV activity. PDTC showed no activity in disrupting the mitogen-activated protein kinase (MAPK) pathway activation induced by viral infection that was vital for the virus's propagation. We found that PDTC modulated cellular ubiquitination and, furthermore, influenced HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced PML stability in the nucleus, resulting in the inhibition of viral gene expression. These results suggested that the antiviral activity of PDTC might be mediated by its dysregulation of the cellular ubiquitin-proteasome system (UPS).

A Novel Selective LSD1/KDM1A Inhibitor Epigenetically Blocks Herpes Simplex Virus Lytic Replication and Reactivation from Latency

ABSTRACTCellular processes requiring access to the DNA genome are regulated by an overlay of epigenetic modifications, including histone modification and chromatin remodeling. Similar to the cellular host, many nuclear DNA viruses that depend upon the host cell’s transcriptional machinery are also subject to the regulatory impact of chromatin assembly and modification. Infection of cells with alphaherpesviruses (herpes simplex virus [HSV] and varicella-zoster virus [VZV]) results in the deposition of nucleosomes bearing repressive histone H3K9 methylation on the viral genome. This repressive state is modulated by the recruitment of a cellular coactivator complex containing the histone H3K9 demethylase LSD1 to the viral immediate-early (IE) gene promoters. Inhibition of the activity of this enzyme results in increased repressive chromatin assembly and suppression of viral gene expression during lytic infection as well as reactivation from latency in a mouse ganglion explant model. However, available small-molecule LSD1 inhibitors are not originally designed to inhibit LSD1, but rather monoamine oxidases (MAO) in general. Thus, their specificity for and potency to LSD1 is low. In this study, a novel specific LSD1 inhibitor was identified that potently repressed HSV IE gene expression, genome replication, and reactivation from latency. Importantly, the inhibitor also suppressed primary infection of HSV in vivo in a mouse model. Based on common control of a number of DNA viruses by epigenetic modulation, it was also demonstrated that this LSD1 inhibitor blocks initial gene expression of the human cytomegalovirus and adenovirus type 5.IMPORTANCE Epigenetic mechanisms, including histone modification and chromatin remodeling, play important regulatory roles in all cellular processes requiring access to the genome. These mechanisms are often altered in disease conditions, including various cancers, and thus represent novel targets for drugs. Similarly, many viral pathogens are regulated by an epigenetic overlay that determines the outcome of infection. Therefore, these epigenetic targets also represent novel antiviral targets. Here, a novel inhibitor was identified with high specificity and potency for the histone demethylase LSD1, a critical component of the herpes simplex virus (HSV) gene expression paradigm. This inhibitor was demonstrated to have potent antiviral potential in both cultured cells and animal models. Thus, in addition to clearly demonstrating the critical role of LSD1 in regulation of HSV infection, as well as other DNA viruses, the data extends the therapeutic potential of chromatin modulation inhibitors from the focused field of oncology to the arena of antiviral agents.

Biology of fowl adenovirus type 1 infection of heterologous cells

The JM1/1 strain of fowl adenovirus (FAV) serotype 1 isolated from gizzard erosion was used to investigate the biology of FAV in homologous (susceptible) and heterologous cells. The FAV JM1/1 strain is capable of efficient multiplication in primary chicken kidney (CK) cells, but not in Crandell-Rees feline kidney (CRFK) cells or Vero cells. FAV adsorption in heterologous cells was slightly higher than in CK cells. An early gene encoding a DNA-binding protein and a late gene encoding the hexon protein were expressed in CK cells. Only the early gene was expressed in Vero cells. Neither of these genes was expressed in CRFK cells. These results suggest that the virus was unable to multiply effectively due to suppression of viral gene expression in the heterologous cells used in this study.

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

In cell cultures, HSV-1 replication is initiated by recruitment by virion protein 16 of transcriptional factors and histone-modifying enzymes to immediate early (α) gene promoters. HSV establishes latent infections characterized by suppression of viral gene expression except for latency-associated transcripts (LATs) and miRNAs. The latent virus reactivates in stressed neurons. A fundamental question is how reactivation initiates in the absence of virion protein 16. We report the following findings in the ganglion explant model. (i) Anti-nerve growth factor antibody accelerated the reactivation of latent virus. Viral mRNAs were detected as early as 9 h after explantation. (ii) After explantation the amounts of viral mRNAs increased whereas amounts of miRNAs and LATs decreased. The decrease in miRNAs and LATs required ongoing protein synthesis, raising the possibility that LAT and miRNAs were degraded by a viral gene product. (iii) The expression of viral genes in explanted ganglia was disordered rather than sequentially ordered as in infected cells in culture. These findings suggest that in reactivating ganglia gene expression is totally derepressed and challenge the current models in that establishment of or exit from latency could not be dependent on the suppression or activation of single or small clusters of viral genes. Finally, miRNAs and LATs reached peak levels 9-11 d after corneal inoculation, thus approximating the pattern of virus replication in these ganglia. These findings suggest that the patterns of accumulation of LATs and miRNAs reflect many different stages in the infection of neurons.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain.

Bovine leukemia virus integration site selection in cattle that develop leukemia

It is essential for efficient replication of retroviruses that the viral genome is integrated into the host genome after reverse transcription. Some retroviruses are preferentially integrated into certain genomic regions that may differ depending on the disease. In this study, we analyzed the integration site of bovine leukemia virus (BLV) in leukemic cells and 55 integration sites were determined. Although the integration sites were not located in a particular chromosome, the BLV provirus was integrated into transcription units at a frequency of 43.6% (24/55) and the transcriptional direction of the provirus was in accordance with that of the integrated host genes in 62.5% (15/24). The integration sites were located in introns of the host gene, excluding only one site, which was located in downstream from a stop codon. BLV provirus was never found in a protein coding sequence (CDS) in this study. Moreover, the BLV provirus did not favor integration near transcription start sites and CpG islands, or repetitive sequences such as transposons. Therefore, the possibility that the integration of the BLV provirus disrupts the host gene is very low. Although a hot spot was not found in the BLV provirus integration sites, the provirus favored the integration into regions disadvantageous for viral gene expression since no integration site was preferentially located into/near CDS, transcription start site or CpG island. It is suggested that the integration site of the BLV provirus in leukemic cells is related to the suppression of viral gene expression.

Salmonella -mediated delivery of RNase P-based ribozymes for inhibition of viral gene expression and replication in human cells

A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87-90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella-based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella-mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.

E box motifs as mediators of proviral latency of human retroviruses

The palindromic sequence motifs (CANNTG) known as E boxes are considered as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. Their presence has been reported in the long terminal repeats (LTR) of the HIV-1 and HTLV-1 proviruses. Their close proximity with the TATA region of both LTRs indicates that the bHLH proteins may act as important regulators of the function of proviral transcription. Indeed, observations on HIV-1 and recent results on HTLV-1 underline that these E boxes may be critically involved in the regulation of the proviral transcription of these human retroviruses. Indeed, of the two E boxes flanking the TATA sequences of the HIV-1 provirus, the 3' E box has been implicated in the transcriptional inhibition of viral gene expression. Such a role might also be played by the unique 5' E box present in the HTLV-1 LTR. In both cases, the expression of tissue-specfic bHLH proteins, like TAL1 might counteract the inhibitory effect exerted by E box proteins, thereby increasing proviral transcription. Finally, a phylogenetic study encompassing several subtypes of these two human retroviruses underlines that these E box motifs have recently appeared in the proviral LTRs and may be considered as potential mediators in the establishment of proviral latency.

Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep

BackgroundDuring malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts.ResultsIn the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303) replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303) had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset.ConclusionOur findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression of viral gene expression is a contributory factor in the impairment of immune surveillance and the uncontrolled proliferation of the BLV-infected tumor cell.