Recombinant baculovirus expressing the FrC-OVA protein induces protective antitumor immunity in an EG7-OVA mouse model.

BACKGROUND: The baculovirus (BV) Autographa californica multiple nuclear polyhedrosis virus has been used in numerous protein expression systems because of its ability to infect insect cells and serves as a useful vaccination vector with several benefits, such as its low clinical risks and posttranslational modification ability. We recently reported that dendritic cells (DCs) infected with BV stimulated antitumor immunity. The recombinant BV (rBV) also strongly stimulated peptide-specific T-cells and antitumor immunity. In this study, the stimulation of an immune response against EG7-OVA tumors in mice by a recombinant baculovirus-based combination vaccine expressing fragment C-ovalbumin (FrC-OVA-BV; rBV) was evaluated. RESULTS: We constructed an rBV expressing fragment C (FrC) of tetanus toxin containing a promiscuous MHC II-binding sequence and a p30-ovalbumin (OVA) peptide that functions in the MHC I pathway. The results showed that rBV activated the CD8+ T-cell-mediated response much more efficiently than the wild-type BV (wtBV). Experiments with EG7-OVA tumor mouse models showed that rBV significantly decreased tumor volume and increased survival compared with those in the wild-type BV or FrC-OVA DNA vaccine groups. In addition, a significant antitumor effect of classic prophylactic or therapeutic vaccinations was observed for rBV against EG7-OVA-induced tumors compared with that in the controls. CONCLUSION: Our findings showed that FrC-OVA-BV (rBV) induced antitumor immunity, paving the way for its use in BV immunotherapy against malignancies.

A 30-year record reveals re-equilibration rates of 137Cs in marine biota after the Fukushima Dai-ichi nuclear power plant accident: Concentration ratios in pre- and post-event conditions.

Concentration ratios (CRs), expressed by dividing 137Cs activity in seawater by that in marine biota (mainly fish), were obtained from the monitoring of 137Cs in coastal areas around Japan between 1984 and 2016. Before the TEPCO Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident (1984-2010), mean CRs of 137Cs, mainly from global fallout (i.e. CRGF), were almost constant for each species throughout the monitoring period, but were different among species, while the values for several species were dependent on their length (i.e. CRGF-SIZE). Thus, CRGF and CRGF-SIZE values for 29 of marketable species are given here as references for conditions where marine biota are in approximate equilibrium (or steady state) with their host water with respect to 137Cs activities in the marine environment. After the FDNPP accident (2011-2016), the impact of the accident has been sustained in eastern Japan waters as indicated by apparent CRs (CRas) which are being used here as indicators of disequilibrium between organisms and their host water. The recession rates of this disequilibrium (the effective CRa half-lives) ranged from 100 to 1100 days. The identified distinct variation was due to the sample locations, even for the same species, because of the change in 137Cs activity concentrations in their host water and diet preference differences. Variation among species, even those captured from the same area, was mainly due to diet differences as well as metabolic-physiological differences in 137Cs retention. Thus, our results from >30 years of systematically monitoring have helped quantify the recession rates of post-FDNPP disequilibrium of 137Cs in biota for assessment of how long term is required from contaminated condition by underlying spatial, inter- and intra-species factors.

[Optimization of Scan Parameters for Patient without Breath Hold in Chest by Computed Tomography].

This study aims to establish optimal scan parameters by high temporal resolution computed tomography (CT) scan for emergency patients who cannot hold their breath. First, we investigated scan parameters that can reduce the effect of motion by evaluating motion artifacts from the moving phantom scan and the temporal sensitivity profile (TPS) measurement. Second, we confirmed the standard deviation (SD) of the CT values as well as the operating time and exposure dose. As the results, plan C [rotation time: 0.275 s, detector rows: 80, pitch factor (PF): 1.100] and plan E (rotation time: 0.275 s, detector rows: 100, PF: 0.880) demonstrated high temporal resolution. The difference between the two is PF. The noise of plan C increased because PF is higher than plan E. This is also evident from the results of SD measurement. Our study demonstrates that the optimal parameters for patients who cannot hold their breath in the emergency care are plan C and plan E. In conclusion, we clarified necessary optimal scan parameters to provide clinical image that has more diagnostic information by reducing the effect of breath motion for emergency patients.

Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG.

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell-cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2.

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation.

Hepatitis C virus utilizes VLDLR as a novel entry pathway.

Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.

A tumor lysate is an effective vaccine antigen for the stimulation of CD4(+) T-cell function and subsequent induction of antitumor immunity mediated by CD8(+) T cells.

To develop a potent cancer vaccine, it is important to study how to prepare highly immunogenic antigens and to identify the most appropriate adjuvants for the antigens. Here we show that a tumor lysate works as an effective antigen to prime CD4(+) T-cell help when baculovirus is employed as an adjuvant. When immunized intradermally with the combination (BLP) of baculovirus, a CT26 tumor lysate, and a cytotoxic T-cell epitope peptide before a tumor challenge, 60% of mice rejected tumors. In contrast, all mice vaccinated with baculovirus plus a tumor lysate (BL) developed tumors. In addition, flow cytometry showed that tumor-specific, interferon γ-producing CD8(+) cytotoxic T lymphocytes (CTLs) were robustly activated by intradermal immunization with BLP. When BLP was administered therapeutically to tumor-bearing mice, antitumor efficacy was better compared to BL. The established tumor was completely eradicated in 50-60% of BLP-treated mice, and induction of tumor-specific CTLs was observed, suggesting that the antitumor efficacy of BLP is mediated by CD8(+) T cells. Numerous CD4(+) T cells infiltrated the tumors of BLP-treated mice, whereas the antitumor effect of BLP almost disappeared after removal of the tumor lysate from BLP or after depletion of BLP-immunized mice of CD4(+) T cells. Thus, the combination of a peptide, lysate, and baculovirus provides stronger antitumor immunity than does a peptide plus baculovirus or a lysate plus baculovirus; effectiveness of BLP is determined by functioning of CD4(+) T cells stimulated with a tumor lysate.

Baculovirus Infection of Human Monocyte-Derived Dendritic Cells Restricts HIV-1 Replication.

Acquired immune deficiency syndrome (AIDS) is mainly caused by infection with human immunodeficiency virus-1 (HIV-1) and still poses a global threat for which we lack a protective or therapeutic vaccine. Dendritic cells (DCs) play a major role in the onset of HIV infection, providing one of the primary sites of HIV replication, and also act as viral reservoirs in vivo. Previous studies have shown that baculovirus (BV) induces strong host immune responses against infections and malignancies. In this study, we infected human monocyte-derived DCs with recombinant BV (AcCAG-gag) and showed that AcCAG-gag-infected human DCs underwent maturation and produced interferon alpha and other proinflammatory cytokines accompanied by increases in the mRNA and protein expression levels of APOBEC3 (A3A, A3F, and A3G), proteins associated with the inhibition of HIV-1 replication. Surprisingly, HIV-1 inhibition is also observed in human DCs infected with a wild-type BV, as determined by the production of inflammatory cytokines, the expression of A3, and a reduction in the p24 level. Our findings outline the mechanism underlying the inhibition of HIV-1 in BV-infected human DCs and pave the way for the use of BV as an effective tool for immunotherapy against HIV-1.

ZNF10 inhibits HIV-1 LTR activity through interaction with NF-κB and Sp1 binding motifs.

Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed ⩾50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense.

A new role for PGA1 in inhibiting hepatitis C virus-IRES-mediated translation by targeting viral translation factors.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.

The RNA-editing enzyme APOBEC1 requires heterogeneous nuclear ribonucleoprotein Q isoform 6 for efficient interaction with interleukin-8 mRNA.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1) is an intestine-specific RNA-binding protein. However, inflammation or exposure to DNA-damaging agents can induce ectopic APOBEC1 expression, which can result in hepatocellular hyperplasia in animal models. To identify its RNA targets, FLAG-tagged APOBEC1 was immunoprecipitated from transfected HuH7.5 hepatocellular carcinoma cells and analyzed using DNA microarrays. The interleukin-8 (IL8) mRNA was the most abundant co-precipitated RNA. Exogenous APOBEC1 expression increased IL8 production by extending the half-life of the IL8 mRNA. A cluster of AU-rich elements in the 3'-UTR of IL8 was essential to the APOBEC1-mediated increase in IL8 production. Notably, IL8 mRNA did not co-immunoprecipitate with APOBEC1 from lysates of other cell types at appreciable levels; therefore, other factors may enhance the association between APOBEC1 and IL8 mRNA in a cell type-specific manner. A yeast two-hybrid analysis and siRNA screen were used to identify proteins that enhance the interaction between APOBEC1 and IL8 mRNA. Heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) was essential to the APOBEC1/IL8 mRNA association in HuH7.5 cells. Of the seven hnRNPQ isoforms, only hnRNPQ6 enabled APOBEC1 to bind to IL8 mRNA when overexpressed in HEK293 cells, which expressed the lowest level of endogenous hnRNPQ6 among the cell types examined. The results of a reporter assay using a luciferase gene fused to the IL8 3'-UTR were consistent with the hypothesis that hnRNPQ6 is required for APOBEC1-enhanced IL8 production. Collectively, these data indicate that hnRNPQ6 promotes the interaction of APOBEC1 with IL8 mRNA and the subsequent increase in IL8 production.

Prominent steatosis with hypermetabolism of the cell line permissive for years of infection with hepatitis C virus.

Most of experiments for HCV infection have been done using lytic infection systems, in which HCV-infected cells inevitably die. Here, to elucidate metabolic alteration in HCV-infected cells in a more stable condition, we established an HCV-persistently-infected cell line, designated as HPI cells. This cell line has displayed prominent steatosis and supported HCV infection for more than 2 years, which is the longest ever reported. It enabled us to analyze metabolism in the HCV-infected cells integrally combining metabolomics and expression arrays. It revealed that rate-limiting enzymes for biosynthesis of cholesterol and fatty acids were up-regulated with actual increase in cholesterol, desmosterol (cholesterol precursor) and pool of fatty acids. Notably, the pentose phosphate pathway was facilitated with marked up-regulation of glucose-6-phosphate dehydrogenase, a rete-limiting enzyme, with actual increase in NADPH. In its downstream, enzymes for purine synthesis were also up-regulated resulting in increase of purine. Contrary to common cancers, the TCA cycle was preferentially facilitated comparing to glycolysis pathway with a marked increase of most of amino acids. Interestingly, some genes controlled by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of antioxidation and metabolism, were constitutively up-regulated in HPI cells. Knockdown of Nrf2 markedly reduced steatosis and HCV infection, indicating that Nrf2 and its target genes play important roles in metabolic alteration and HCV infection. In conclusion, HPI cell is a bona fide HCV-persistently-infected cell line supporting HCV infection for years. This cell line sustained prominent steatosis in a hypermetabolic status producing various metabolites. Therefore, HPI cell is a potent research tool not only for persistent HCV infection but also for liver metabolism, overcoming drawbacks of the lytic infection systems.

Intradermal immunization with combined baculovirus and tumor cell lysate induces effective antitumor immunity in mice.

Although tumor lysate contains all the potential helper and killer epitopes capable of stimulating T cells, it is difficult to use as a cancer vaccine because it suppresses dendritic cell (DC) function. We report that wild-type baculovirus possesses an adjuvant effect to improve the immunogenicity of tumor lysate. When mice were administered CT26 tumor cell lysate combined with baculovirus intradermally, antitumor immunity was induced and rejection of CT26 tumor growth was observed in 40% of the immunized mice. In contrast, such antitumor immunity was not elicited in mice inoculated with tumor cell lysate or baculovirus alone. In tumor-bearing mice, which had previously received the combined baculovirus and tumor lysate vaccine, the established tumors were completely eradicated by administering a booster dose of the combined vaccine. This antitumor effect was attributed to tumor-specific T cell immunity mediated primarily by CD8⁺ T cells. Baculovirus also strongly activated DCs loaded with tumor lysate. Increased interleukin (IL)-6 and IL-12p70 production were also observed in DCs co-cultured with tumor cell lysate and baculovirus. Our study demonstrates that combined baculovirus and tumor lysate vaccine can effectively stimulate DCs to induce acquired antitumor immunity.

Cyclosporin A inhibits the propagation of influenza virus by interfering with a late event in the virus life cycle.

Influenza is a global public health problem that causes a serious respiratory disease. Influenza virus frequently undergoes amino acid substitutions, which result in the emergence of drug-resistant viruses. To control influenza viruses that are resistant to currently available drugs, it is essential to develop new antiviral drugs with a novel molecular target. Here, we report that cyclosporin A (CsA) inhibits the propagation of influenza virus in A549 cells by interfering with a late event in the virus life cycle. CsA did not affect adsorption, internalization, viral RNA replication, or synthesis of viral proteins in A549 cells, but inhibited the step(s) after viral protein synthesis, such as assembly or budding. In addition, siRNA-mediated knockdown of the expression of the major CsA targets, namely cyclophilin A (CypA), cyclophilin B (CypB), and P-glycoprotein (Pgp), did not inhibit influenza virus propagation. These results suggest that CsA inhibits virus propagation by mechanism(s) independent of the inhibition of the function of CypA, CypB, and Pgp. CsA may target an unknown molecule that works as a positive regulator in the propagation of influenza virus. Our findings would contribute to the development of a novel anti-influenza virus therapy and clarification of the regulatory mechanism of influenza virus multiplication.

Induction of heat-shock protein 70 by prostaglandin A1 inhibits HIV-1 Vif-mediated degradation of APOBEC3G.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A1 (PGA1) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin-proteasome pathway, we examined the effects of PGA1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA1-treated 293T cells. The induction of HSP70 synthesis by PGA1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA1. Thus, we showed that PGA1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

Hepatitis C virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells.

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1ß. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) ß mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPß target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPß in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

High yield production of influenza virus in Madin Darby canine kidney (MDCK) cells with stable knockdown of IRF7.

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/ß induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.

Inhibition of HIV-1 replication by RNA with a microRNA-like function.

Human immunodeficiency virus type 1 (HIV-1) replication is suppressed by a small guide RNA (sgRNA) that targets the packaging signal of HIV-1 RNA. We unintentionally produced a plasmid with the reverse sequence of the sgRNA and its terminator (pR-Ψ-sgRNA-ter). Both sgRNA and R-Ψ-sgRNA suppress HIV-1, but the mechanism by which R-Ψ-sgRNA suppresses HIV is not clear. To evaluate whether the suppressive effect is caused by an RNA interference or microRNA (miRNA)-like mechanism, R-Ψ-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important enzyme for RNA interference and miRNA. The RNA was cleaved into fragments of approximately 24 nucleotides (nt). We analyzed the sequence of the RNA fragments and predicted the RNA secondary structure of R-Ψ-sgRNA to determine the region recognized by the Dicer enzyme. The lengths of the R-Ψ-sgRNA fragments ranged from 48 to 140 nt, and were predicted to form double strands, including mismatches, in this region. An HIV-1 p24 assay indicated that the R-Ψ-sgRNA fragments suppressed HIV-1 replication. These findings suggest that R-Ψ-sgRNA acts as a miRNA to inhibit HIV-1.

HIV-1 suppressive sequences are modulated by Rev transport of unspliced RNA and are required for efficient HIV-1 production.

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3' untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3' untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.

ZBRK1 represses HIV-1 LTR-mediated transcription.

The identification of cellular proteins that interact with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) provides a basic understanding of HIV-1 gene expression, which is the major determinant regulating virus replication. We show that ZBRK1 negatively regulates the HIV-1 LTR. Ectopic expression of ZBRK1 represses transcriptional activity of the HIV-1 LTR, whereas the depletion of endogenous ZBRK1 leads to activation of the HIV-1 LTR. The repressor activity of ZBRK1 is required for TRIM28 binding. Furthermore, ZBRK1 is bound to the HIV-1 LTR in vivo. These results indicate that ZBRK1 could be involved in a potent intrinsic antiretroviral defense.