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Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.

Gene expression in HIV-1 is regulated by the promoters in 5' long-terminal repeat (LTR) element, which contain multiple DNA regulatory elements that serve as binding sites for cellular transcription factors. YY1 could repress HIV-1 gene expression and latent infection. Here, however, we observed that virus production can be increased by YY1 over-expression and decreased under YY1 depleted condition by siRNA treatment. To identify functional domain(s) of YY1 activation, we constructed a number of YY1 truncated mutants. Our data show that full-length YY1 enhances the viral transcription both through U3 and U3RU5 promoters. Moreover, the C-terminal region (296-414 residues) of YY1 is responsible for the transcriptional upregulation, which could be enhanced further in the presence of the viral Tat protein. The central domain of YY1 (155-295 residues) does not affect LTR activity but has a negative effect on HIV-1 gene expression. Taken together, our study shows that YY1 could act as a transcriptional activator in HIV-1 replication, at least in the early stages of infection. [BMB Reports 2020; 53(5): 248-253].

The positive transcription elongation factor b (P-TEFb) is an essential factor that induces transcription elongation and is also negatively regulated by the cellular factor HEXIM1. Previously, the chimeric protein HEXIM1-Tat (HT) was demonstrated to inhibit human immunodeficiency virus-1 (HIV)-1 transcription. In this study, we attempted to develop an improved antiviral protein that specifically binds viral RNA (vRNA) by fusing HT to HIV-1 nucleocapsid (NC). Thus, we synthesized NC-HEXIM1-Tat (NHT) and HEXIM1-Tat-NC (HTN). NHT and HTN inhibited virus proliferation more effectively than HT, and they did not attenuate the function of HT. Notably, NHT and HTN inhibited the infectivity of the progeny virus, whereas HT had no such effect. NHT and HTN selectively and effectively interacted with vRNA and inhibited the proper packaging of the HIV-1 genome. Taken together, our results illustrated that the novel NC-fused chimeric proteins NHT and HTN display novel mechanisms of anti-HIV effects by inhibiting both HIV-1 transcription and packaging.

In this study, we investigated how Staufen1 influences the HIV-1 production. The overexpression of Staufen1 increased virus production without any negative affect on the viral infectivity. This increase was not caused by transcriptional activation; but by influencing post-transcriptional steps. Using multiple Gag protein derivatives, we confirmed that the zinc-finger domains of the HIV-1 nucleocapsid (NC) are important for its interaction with Staufen1. We also found that Staufen1 colocalized in stress granules with the mature form of the HIV-1 NC protein. [BMB Reports 2021; 54(11): 551-556].

The activating transcription factor (ATF) 4 belongs to the ATF/CREB (cAMP Response Element Binding bZIP [Basic Leucine Zipper]) transcription factor family, and plays a central role in the UPR (Unfolded Protein Response) process in cells. The induction of ATF4 expression has previously been shown to increase the replication of HIV-1. However, the detailed mechanism underlying this effect and the factors involved in the regulation of ATF4 function are still unknown. Here, we demonstrate first that knocking out ATF4 using siRNA shows a strong negative effect on HIV-1 production, indicating that ATF4 is a functional positive cellular factor in HIV-1 production. To determine the mechanism by which ATF4 regulates the HIV-1 life cycle, we assessed the effect of the overexpression of wild type ATF4 and its various derivatives on HIV-1 LTR-mediated transcriptional activation and the production of HIV-1 particles. This effect was studied through co-transfection experiments with either reporter vectors or proviral DNA. We found that the N-terminal domains of ATF4 are involved in HIV-1 LTR-mediated transcriptional activation, and thus in HIV-1 production. [BMB Reports 2018; 51(8): 388-393].

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity. [BMB Reports 2018; 51(7): 338-343].

Y-box binding protein 1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily. It participates in a wide variety of cellular events, including transcription, RNA splicing, translation, DNA repair, drug resistance, and stress responses. We investigated putative functions of YB-1 in HIV-1 replication. Functional studies using overexpression or knockdown of YB-1 in conjunction with transfection of proviral DNA showed that YB-1 enhances virus production. We found YB-1 regulates HIV-1 production by stimulating viral transcription using HIV-1 LTR sequence U3RU5 with Luciferase assay. We also identified a specific region from amino acids 1 to 324 of YB-1 as necessary for the participation of the protein in the production of virions. [BMB Reports 2018; 51(6): 290-295].

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity.

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The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is a multifunctional, zinc finger-containing protein known to be involved in almost every step of the viral life cycle. We therefore examined the effects of NC in vivo as a transcription activator on the basal transcriptional activity of the HIV-1 U3 and Rous sarcoma virus (RSV) promoters, as well as HIV-1 long terminal repeats (LTRs) such as the U3R and U3RU5 regions, using promoter-fused reporter gene assays, Western blot analyses, and quantitative real time-polymerase chain reaction. From these studies, we found that the basal transcriptional levels of the HIV-1 U3 and RSV promoters were barely enhanced by the presence of NC. Placing the U3R region upstream of reporter genes greatly increased transcriptional activity compared to that of the U3 promoter alone, and such activity was further increased by Tat expression. However, neither transcription driven by U3R itself nor Tat-mediated transcriptional activation of the U3R was further increased by the addition of NC. Similar results were also observed with U3RU5 of the HIV-1 LTR region in the presence of either NC or Gag protein. Thus, these results indicate that the HIV NC protein is unable to act as a transcriptional activator on its cognate and possibly other retroviral promoters.

－ Hepatitis C virus (HCV) has genetic diversity like most of RNA viruses. HCV major genotypes are classified into several subtypes which are further divided into quasispecies having, genetically different but closely related variants. The HCV core that is a nucleocapsid protein located at the amino terminus of the viral polyprotein is relatively a conserved protein among the HCV isolates and thus it has been one of plausible targets for anti-HCV drug development. However, different quasispecies of HCV core gene have also been found. In this study, we compared the expression level of core protein between two different quasispecies of HCV genotype 1b. Our data demonstrate that a little differences of amino acid sequence lead to substantial difference of expression level. It might be another important reason of different pathogenesis among HCV infected patients.Keywords: Hepatitis C virus, Core protein, Quasispecies INTRODUCTION Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver transplants and over the 170 million per-sons chronically are infected with it world widely. It is a en-veloped virus with a 9.6 kb positive-sense single-stranded RNA genome encoding a single open reading frame (ORF), which is processed by cellular and viral proteases to produce at least 10 structural and nonstructural proteins. It has been known now that there are at least six major genotypes and a number of subtypes of HCV which are generated due to high error rates in the viral RNA repli-cation, (Choo

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells.

Here we report a new chemical inhibitor against HIV-1 with a novel structure and mode of action. The inhibitor, designated as A1836, inhibited HIV-1 replication and virus production with a 50% inhibitory concentration (IC50) of 2.0 μM in an MT-4 cell-based and cytopathic protection antiviral assay, while its 50% cytotoxic concentration (CC50) was much higher than 50 μM. Examination of the effect of A1836 on in vitro HIV-1 reverse transcriptase (RT) and integrase showed that neither were molecular targets of A1836. The characterization and re-infection assay of the HIV-1 virions generated in the presence of A1836 showed that the synthesis of early RT products in the cells infected with the virions was inhibited dose-dependently, due in part to abnormal protein formation within the virions, thus resulting in an impaired infectivity. These results suggest that A1836 might be a novel candidate for the development of a new type of HIV-1 inhibitor. [BMB Reports 2015; 48(2): 121-126]

Background The human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) is an essential and multifunctional protein involved in multiple stages of the viral life cycle such as reverse transcription, integration of proviral DNA, and especially genome RNA packaging. For this reason, it has been considered as an attractive target for the development of new anti-HIV drugs. Although a number of inhibitors of NC have been reported thus far, the search for NC-specific and functional inhibitor(s) with a good antiviral activity continues. Results In this study, we report the identification of A1752, a small molecule with inhibitory action against HIV-1 NC, which shows a strong antiviral efficacy and an IC50 around 1 μM. A1752 binds directly to HIV-1 NC, thereby inhibiting specific chaperone functions of NC including Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization, and it also disrupts the proper Gag processing. Further analysis of the mechanisms of action of A1752 also showed that it generates noninfectious viral particles with defects in uncoating and reverse transcription in the infected cells. Conclusions These results demonstrate that A1752 is a specific and functional inhibitor of NC with a novel mode of action and good antiviral efficacy. Thus, this agent provides a new type of anti-HIV NC inhibitor candidate for further drug development. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0218-9) contains supplementary material, which is available to authorized users.