The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

A novel class III endogenous retrovirus with a class I envelope gene in African frogs with an intact genome and developmentally regulated transcripts in Xenopus tropicalis.

BACKGROUND: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. RESULTS: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. CONCLUSIONS: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.

Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase.

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.

Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV.

HIV has a high mutation rate, which contributes to its ability to evolve quickly. However, we know little about the fitness costs of individual HIV mutations in vivo, their distribution and the different factors shaping the viral fitness landscape. We calculated the mean frequency of transition mutations at 870 sites of the pol gene in 160 patients, allowing us to determine the cost of these mutations. As expected, we found high costs for non-synonymous and nonsense mutations as compared to synonymous mutations. In addition, we found that non-synonymous mutations that lead to drastic amino acid changes are twice as costly as those that do not and mutations that create new CpG dinucleotides are also twice as costly as those that do not. We also found that G→A and C→T mutations are more costly than A→G mutations. We anticipate that our new in vivo frequency-based approach will provide insights into the fitness landscape and evolvability of not only HIV, but a variety of microbes.

Hepatitis B virus-triggered PTEN/ß-catenin/c-Myc signaling enhances PD-L1 expression to promote immune evasion.

Hepatitis B virus (HBV) exploits multiple strategies to evade host immune surveillance. Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling plays a critical role in regulating T cell homeostasis. However, it remains largely unknown as to how HBV infection elevates PD-L1 expression in hepatocytes. A mouse model of HBV infection was established by hydrodynamic injection with a vector containing 1.3-fold overlength HBV genome (pHBV1.3) via the tail vein. Coculture experiments with HBV-expressing hepatoma cells and Jurkat T cells were established in vitro. We observed significant decrease in the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and increase in ß-catenin/PD-L1 expression in liver tissues from patients with chronic hepatitis B and mice subjected to pHBV1.3 hydrodynamic injection. Mechanistically, decrease in PTEN enhanced ß-catenin/c-Myc signaling and PD-L1 expression in HBV-expressing hepatoma cells, which in turn augmented PD-1 expression, lowered IL-2 secretion, and induced T cell apoptosis. However, ß-catenin disruption inhibited PTEN-mediated PD-L1 expression, which was accompanied by decreased PD-1 expression, and increased IL-2 production in T cells. Luciferase reporter assays revealed that c-Myc stimulated transcriptional activity of PD-L1. In addition, HBV X protein (HBx) and HBV polymerase (HBp) contributed to PTEN downregulation and ß-catenin/PD-L1 upregulation. Strikingly, PTEN overexpression in hepatocytes inhibited ß-catenin/PD-L1 signaling and promoted HBV clearance in vivo. Our findings suggest that HBV-triggered PTEN/ß-catenin/c-Myc signaling via HBx and HBp enhances PD-L1 expression, leading to inhibition of T cell response, and promotes HBV immune evasion.NEW & NOTEWORTHY This study demonstrates that during HBV infection, HBV can increase PD-L1 expression via PTEN/ß-catenin/c-Myc signaling pathway, which in turn inhibits T cell response and ultimately promotes HBV immune evasion. Targeting this signaling pathway is a potential strategy for immunotherapy of chronic hepatitis B.

Novel Modified Vaccinia Virus Ankara Vector Expressing Anti-apoptotic Gene B13R Delays Apoptosis and Enhances Humoral Responses.

Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+ DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

Association of HBsAg mutation patterns with hepatitis B infection outcome: Asymptomatic carriers versus HCC/cirrhotic patients.

INTRODUCTION AND OBJECTIVES: The hepatitis B virus (HBV) surface antigen (HBsAg) variations suggested having some effects on infection outcome. Due to some controversial issues, the aim of this study was to compare the pattern of HBsAg variation between asymptomatic carriers and HCC/cirrhosis patients. MATERIALS AND METHODS: In this cross-sectional study, 19 HCC/cirrhotic and 26 asymptomatic patients were enrolled. After viral DNA extraction, HBs gene was amplified using an in-house nested-PCR. Then, PCR products were introduced into bi-directional Sanger sequencing. The retrieved sequences were compared with references, to investigate the variation of immunologic sites, major hydrophilic region (MHR) of HBsAg as well as reverse transcriptase (RT), and also to determine genotype/subtype. RESULTS: The analysis of MHR and epitopes on HBsAg showed dozens of substitution, which occurred more prevalently in I110, P120, Y134, G159, S193, Y206, S207, I208, L213 and P214 positions. However, Y134N/F/L (P=0.04) and P120T/S (P=0.009) were significantly detected in MHR and B-cell epitope of HCC/Cirrhotic group. A number of truncation-related mutations were higher in HCC/Cirrhotic group (P>0.001), albeit only C69* stop codon was statistically significant (P=0.003). In RT, some potentially resistant substitutions such as Q215S, V191I and V214A, were revealed. Phylogenetic analysis showed that all of isolates belonged to genotype D, and the major serotype was ayw1. CONCLUSION: The higher frequency of substitutions in MHR and immune epitopes at positions such as Y134 and P120 as well as stop codons such as C69* in HCC/cirrhotic group might candidate them as predictive factors for infection outcome.

[HBV pol/S gene mutations in chronic hepatitis B patients receiving nucleoside/nucleotide analogues treatment]. / Nükleozid/nükleotid analogu tedavisi alan kronik hepatit B hastalarinda hbv pol/S geni mutasyonlari.

Chronic hepatitis B (CHB) is an important public health problem affecting over 240 million people all around the world. The aim of the treatment in chronic hepatitis B is to prevent progression to cirrhosis and liver cancer. Interferons (standard and peginterferon) (Peg-IFN) and nucleoside/nucleotide analogues (NAs) are widely used in the treatment of CHB. The use of long-term therapy can however result in drug resistant mutations, which can lead to treatment failure. In patients with chronic hepatitis B, in addition to primary drug resistance mutations in the pol gene, compensatory mutations were reported. The genom of HBV polymerase (pol) gene overlaps with the envelope (S) gene. Nucleoside/nucleotide analogue (NA) resistance mutations in the pol gene of HBV, either from selection of primary or secondary resistance mutations, typically result in changes in HBsAg. Recent studies have conferred a new acronym for these HBV pol/S gene overlap mutants; ADAPVEMs, for antiviral drug-associated potential vaccine-escape mutants. The aim of this study was to investigate clinically and epidemiologically significant HBV pol/S gene mutations in NA treated CHB patients. In the study, a total of 100 patients who received nucleoside/nucleotide analogue therapy for one year or more were included. The levels of HBV DNA from serum samples were detected by the commercial real-time PCR assay and the mutations of pol/S genes by direct sequencing. Sixteen samples with low HBV DNA levels (> 200 IU/ml) could not be interpreted by sequencing due to insufficient amplification. Of the remaining 84 patients that could be sequenced HBV pol gene of HBV, 53 (63.09%) were males and 31 (36.91%) were women and the mean age was 47 ± 14.99 years (range: 20-67). Primary/secondary drug mutations (rtM204I/V, rtI169S, rtL180M, rtT184L, rtA194V, rtM204I/rtL91I, rtQ149K, rtQ215H/S, rtN238D) were detected in 38 (45.2%) of the patients. Because of the HBV pol/S gene overlapping, in 27 patients immun-selected amino acid substitutions (sI110L, sT127P, sS114A, sT123A), in nine patients HBIg selected escape mutants (sP120R, sT123N, sE164D, sY134F, sQ129H, sT118A, sP127K), in seven patients vaccine escape mutants (sT126I, sP120S, sG145A, s S193L) and in one patient misdiagnosis of HBsAg (sT131I) were detected. In addition, antiviral drug-associated potential vaccine-escape mutants were detected in 13 (15.4%) patients. In patients with chronic HBV, NAs including commonly used lamivudine were observed to have the potential for ADAPVEM to emerge during treatment. It was concluded that after determination of antiviral drug resistance and ADAPVEMs replanning of treatment should be done in the NA treatment of patients with CHB.

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.

Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain. IMPORTANCE: HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

Is HERV-K and HERV-W Expression Regulated by miR-155 in Kidney Transplant Patients with Human Cytomegalovirus Infection?

According to the latest update, 2,578 unique mature micro-RNAs (miRNAs) are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high human endogenous retrovirus, HERV, K vs. low) showed that 2 miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and miR-155 expression in kidney transplant recipients and the possible relationship between them. The comparison between kidney transplant patients negative for human cytomegalovirus (HCMV) infection and positive patients showed a significant difference in terms of miR-155 expression (p = 0.0111). We demonstrated that HERV-K and -W pol gene expression was significantly higher in CMV-infected kidney transplant recipients versus those not infected as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERV notwithstanding that we together observed increased expression of HERV-K and -W and diminished expression of miR-155 in HCMV-infected human kidney transplant recipients.

Isolation and sequence analysis of a novel rhesus macaque foamy virus isolate with a serotype-1-like env.

SFVmmu-DPZ9524 represents the third completely sequenced rhesus macaque simian foamy virus (SFV) isolate, alongside SFVmmu_K3T with a similar SFV-1-type env, and R289HybAGM with a SFV-2-like env. Sequence analysis demonstrates that, in gag and pol, SFVmmu-DPZ9524 is more closely related to R289HybAGM than to SFVmmu_K3T, which, outside of env, is more similar to a Japanese macaque isolate than to the other two rhesus macaque isolates SFVmmu-DPZ9524 and R289HybAGM. Further, we identify bel as another recombinant locus in R289HybAGM, confirming that recombination contributes to sequence diversity in SFV.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy.

BACKGROUND & AIMS: Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). METHODS: Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). RESULTS: Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. CONCLUSIONS: The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. LAY SUMMARY: Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.

CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees.

Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals.

Prevalence of Antiretroviral Drug Resistance in Patients Who Are Not Responding to Protease Inhibitor-Based Treatment: Results From the First National Survey in South Africa.

Limited data exist on human immunodeficiency virus type 1 (HIV-1) resistance in patients who are not responding to protease inhibitor (PI)-based regimens in resource-limited settings. This study assessed resistance profiles in adults across South Africa who were not responding to PI-based regimens. pol sequencing was undertaken and submitted to the Stanford HIV Drug Resistance Database. At least 1 major PI mutation was detected in 16.4% of 350 participants. A total of 53.4% showed intermediate resistance to darunavir/ritonavir, whereas high-level resistance was not observed. Only 5.2% and 32.8% of participants showed high-level and intermediate resistance to etravirine, respectively. Although the prevalence of major PI mutations was within previously reported ranges, most patients will likely experience virological suppression during receipt of currently available South African third-line regimens.

Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency.

BACKGROUND: While antiretroviral therapies have improved life expectancy and reduced viral loads in HIV-1-positive individuals, the cessation of treatment results in a rebound of viral replication. This suggests that a reservoir of latently-infected cells remains within these patients, the identity of which is ill-defined and therefore difficult to target therapeutically. Current strategies are aimed at using drugs such as histone deacetylase (HDAC) inhibitors to induce the expression of latent HIV-1 proviruses in order to activate and ultimately eradicate this reservoir of infected cells. One concern with the use of HDAC inhibitors is that they could up-regulate human endogenous retroviruses (HERVs), as well as HIV-1, with potentially pathophysiological consequences. RESULTS: In this study, we analysed the transcription of HERV genes in HIV-1 latency T cell (J-LAT 8.4) and monocyte (U1) models following treatment with the HDAC inhibitors, vorinostat, panobinostat and romidepsin. We examined the expression of HERV-K (HML-2) env and pol, as well as the co-opted genes HERV-W env (syncytin-1), HERV-FRD env (syncytin-2), in these cell lines. Finally, we investigated HERV expression in primary human T cells. CONCLUSIONS: We show that HDAC inhibitors did not substantially increase the transcription of the analysed HERV env or pol genes, suggesting that histone acetylation is not crucial for controlling HERV expression in these experimental models and in ex vivo primary human T cells. Importantly, this indicates that unwanted HERV expression does not appear to be a barrier to the use of HDAC inhibitors in HIV-1 cure strategies.

Time dependency of foamy virus evolutionary rate estimates.

BACKGROUND: It appears that substitution rate estimates co-vary very strongly with their timescale of measurement; the shorter the timescale, the higher the estimated value. Foamy viruses have a long history of co-speciation with their hosts, and one of the lowest estimated rates of evolution among viruses. However, when their rate of evolution is estimated over short timescales, it is more reminiscent of the rapid rates seen in other RNA viruses. This discrepancy between their short-term and long-term rates could be explained by the time-dependency of substitution rate estimates. Several empirical models have been proposed and used to correct for the time-dependent rate phenomenon (TDRP), such as a vertically-translated exponential rate decay model and a power-law rate decay model. Nevertheless, at present, it is still unclear which model best describes the rate dynamics. Here, we use foamy viruses as a case study to empirically describe the phenomenon and to determine how to correct rate estimates for its effects. Four empirical models were investigated: (i) a vertically-translated exponential rate decay model, (ii) a simple exponential rate decay model, (iii) a vertically-translated power-law rate decay model, and (iv) a simple power-law rate decay model. RESULTS: Our results suggest that the TDRP is likely responsible for the large discrepancy observed in foamy virus short-term and long-term rate estimates, and the simple power-law rate decay model is the best model for inferring evolutionary timescales. Furthermore, we demonstrated that, within the Bayesian phylogenetic framework, currently available molecular clocks can severely bias evolutionary date estimates, indicating that they are inadequate for correcting for the TDRP. Our analyses also suggest that different viral lineages may have different TDRP dynamics, and this may bias date estimates if it is unaccounted for. CONCLUSIONS: As evolutionary rate estimates are dependent on their measurement timescales, their values must be used and interpreted under the context of the timescale of rate estimation. Extrapolating rate estimates across large timescales for evolutionary inferences can severely bias the outcomes. Given that the TDRP is widespread in nature but has been noted only recently the estimated timescales of many viruses may need to be reconsidered and re-estimated. Our models could be used as a guideline to further improve current phylogenetic inference tools.

Different patterns of codon usage in the overlapping polymerase and surface genes of hepatitis B virus suggest a de novo origin by modular evolution.

The polymerase (P) and surface (S) genes of hepatitis B virus (HBV) show the longest gene overlap in animal viruses. Gene overlaps originate by the overprinting of a novel frame onto an ancestral pre-existing frame. Identifying which frame is ancestral and which frame is de novo (the genealogy of the overlap) is an appealing topic. However, the P/S overlap of HBV is an intriguing paradox, because both genes are indispensable for virus survival. Thus, the hypothesis of a primordial virus without the surface protein or without the polymerase makes no biological sense. With the aim to determine the genealogy of the overlap, the codon usage of the overlapping frames P and S was compared to that of the non-overlapping region. It was found that the overlap of human HBV had two patterns of codon usage. One was localized in the 59 one-third of the overlap and the other in the 39 two-thirds. By extending the analysis to non-human HBVs, it was found that this feature occurred in all hepadnaviruses. Under the assumption that the ancestral frame has a codon usage significantly closer to that of the non-overlapping region than the de novo frame, the ancestral frames in the 59 and 39 region of the overlap could be predicted. They were, respectively, frame S and frame P. These results suggest that the spacer domain of the polymerase and the S domain of the surface protein originated de novo by overprinting. They support a modular evolution hypothesis for the origin of the overlap.

Synergistic inhibition of avian leukosis virus subgroup J replication by miRNA-embedded siRNA interference of double-target.

BACKGROUND: The diseases caused by avian leukosis virus subgroup J (ALV-J) has become a serious problem in the poultry. Due to largely ineffective vaccines, new control measures are needed to be developed. RNA interference (RNAi) has been developed a promising measure for antivirus in poultry. METHODS: In this study, miRNA-embedded siRNA interference was designed and used to inhibit ALV-J replication in vitro and in vivo. Each sequence of target siRNA derived from the gag (p15), pol (p32), env (gp85) and LTR (U3) gene of ALV-J was embedded into mouse miR-155 backbone as a pre-miRNA hairpin oligonucleotide sequence. After annealing, they were cloned into pcDNA6.2-GW/EmGFP-miR vector, respectively. For detecting the interference effect, recombinant vectors were introduced into DF-1 cells and day-old SPF chickens that infected with ALV-J. RESULTS: In vitro, single target interference showed effective inhibition of reducing 74% ~ 85% mRNA of ALV-J. Double targets showed more efficient inhibition of reducing 96% ~ 98% mRNA of ALV-J. In vivo, chicks were inoculated with each recombinant plasmid in peritoneal cavity at day of hatch, and monitored infection status at interval 1 day postinfection for 4 weeks. Delivery of single target or double targets miRNA significantly reduced viremia and pathogenicity caused by ALV-J in vivo, especially the double targets. CONCLUSIONS: These data demonstrated that the miRNA-embedded siRNA interference is an efficient method for inhibition of ALV-J replication, especially double targets.

Delayed-onset enzootic bovine leukosis possibly caused by superinfection with bovine leukemia virus mutated in the pol gene.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), to which animals are most susceptible at 4-8 years of age. In this study, we examined tumor cells associated with EBL in an 18-year-old cow to reveal that the cells carried at least two different copies of the virus, one of which was predicted to encode a reverse transcriptase (RT) lacking ribonuclease H activity and no integrase. Such a deficient enzyme may exhibit a dominant negative effect on the wild-type RT and cause insufficient viral replication, resulting in delayed tumor development in this cow.