HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

Successful selective production of vanillic acid from depolymerized sulfite lignin and its application to poly(ethylene vanillate) synthesis.

Towards lignin upgrading, vanillic acid (VA), a lignin-derived guaiacyl compound, was produced from sulfite lignin for successfully synthesizing poly(ethylene vanillate), an aromatic polymer. The engineered Sphingobium sp. SYK-6-based strain in which the genes responsible for VA/3-O-methyl gallic acid O-demethylase and syringic acid O-demethylase were disrupted was able to produce vanillic acid (VA) from the mixture consisting of acetovanillone, vanillin, VA, and other low-molecular-weight aromatics obtained by Cu(OH)2-catalyzed alkaline depolymerization of sulfite lignin and membrane fractionation. From the bio-based VA, methyl-4-(2-hydroxyethoxy)-3-methoxybenzoate was synthesized via methylesterification, hydroxyethylation, and distillation, and then it was subjected to polymerization catalyzed by titanium tetraisopropoxide. The molecular weight of the obtained poly(ethylene vanillate) was evaluated to be Mw = 13,000 (Mw/Mn = 1.99) and its melting point was 261 °C. The present work proved that poly(ethylene vanillate) is able to be synthesized using VA produced from lignin for the first time.

Vaccination with short-term-cultured autologous PBMCs efficiently activated STLV-1-specific CTLs in naturally STLV-1-infected Japanese monkeys with impaired CTL responses.

A small proportion of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma, a chemotherapy-resistant lymphoproliferative disease with a poor prognosis. HTLV-1-specific cytotoxic T lymphocytes (CTLs), potential anti-tumor/virus effectors, are impaired in adult T-cell leukemia/lymphoma patients. Here, using Japanese monkeys naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) as a model, we demonstrate that short-term-cultured autologous peripheral blood mononuclear cells (PBMCs) can serve as a therapeutic vaccine to activate such CTLs. In a screening test, STLV-1-specific CTL activity was detectable in 8/10 naturally STLV-1-infected monkeys. We conducted a vaccine study in the remaining two monkeys with impaired CTL responses. The short-term-cultured PBMCs of these monkeys spontaneously expressed viral antigens, in a similar way to PBMCs from human HTLV-1 carriers. The first monkey was subcutaneously inoculated with three-day-cultured and mitomycin C (MMC)-treated autologous PBMCs, and then boosted with MMC-treated autologous STLV-1-infected cell line cells. The second monkey was inoculated with autologous PBMC-vaccine alone twice. In addition, a third monkey that originally showed a weak STLV-1-specific CTL response was inoculated with similar autologous PBMC-vaccines. In all three vaccinated monkeys, marked activation of STLV-1-specific CTLs and a mild reduction in the STLV-1 proviral load were observed. Follow-up analyses on the two monkeys vaccinated with PBMCs alone indicated that STLV-1-specific CTL responses peaked at 3-4 months after vaccination, and then diminished but remained detectable for more than one year. The significant reduction in the proviral load and the control of viral expression were associated with CTL activation but also diminished 6 and 12 months after vaccination, respectively, suggesting the requirement for a booster. The vaccine-induced CTLs in these monkeys recognized epitopes in the STLV-1 Tax and/or Envelope proteins, and efficiently killed autologous STLV-1-infected cells in vitro. These findings indicated that the autologous PBMC-based vaccine could induce functional STLV-1-specific CTLs in vivo.

Antifungal activity of simply fractionated organosolv lignin against Trametes versicolor.

In an effort to achieve sustainable development goals, a reevaluation of the materials used in wooden buildings must be done, including the preservatives used to treat the materials. Since typical wood preservatives use toxic heavy metals, their handling and use can contaminate the environment. Therefore, substances such as lignin-derived components have been investigated as bio-based preservatives. Organosolv treatment is a promising technique for separating components of lignocellulosic biomass, which enables the utilization of each component. The present report describes components of lignocellulose with antifungal effects that were recovered after organosolv treatment using water and 1-butanol solvent at 473 K for 2 h, followed by simple solvent fractionation. The organosolv lignin was divided into three fractions: n-hexane soluble, ethyl acetate soluble, and ethyl acetate insoluble, yielding 23 wt%, 52 wt% and 13 wt%, respectively. Antifungal activity was determined using an agar plate method. White rot fungi (Trametes versicolor) was dispersed on the agar plate with a cellulose disc containing each lignin-derived fraction obtained from Japanese cedar. Results showed inhibition of fungal growth over the cellulose disc containing the n-hexane soluble fraction. To examine the effect in greater detail, the chemical structure of the n-hexane-soluble fraction on the antifungal activity was investigated. The content of phenolic hydroxyl group in n-hexane-soluble fraction was the highest (4.6 mmol/g), and the results from the chemical modification suggested that the functional group was required for antifungal action. In addition, the n-hexane-soluble fraction imparted some water resistance. The procedures used for cedar as a feedstock were applied to another type of biomass-bagasse-and its fractions showed antifungal activity similar to those of Japanese cedar.

Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase.

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN's effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 µM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.

Involvement of EZH2 inhibition in lenalidomide and pomalidomide-mediated growth suppression in HTLV-1-infected cells.

Immunomodulatory imide drugs (IMiDs), such as lenalidomide and pomalidomide, exert pleiotropic effects, e.g., antitumor effects in multiple myeloma, by binding the protein Cereblon and altering its substrate specificity. Lenalidomide is approved for the treatment of adult T-cell leukemia/lymphoma (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1), although the precise mechanisms responsible for its effectiveness have not been fully elucidated. Here, we used HTLV-1-infected cell lines to investigate how IMiDs exert anti-ATL effects. In three of four tested HTLV-1-infected cell lines, the cells treated with lenalidomide or pomalidomide exhibited mild growth suppression without apoptosis, which was associated with decreased IRF4, c-Myc, and phosphorylated STAT3 levels as well as enhanced SOCS3 expression. Additionally, the levels of enhancer of zeste homolog 2 (EZH2) and trimethyl histone 3 Lys27 (H3K27me3) were decreased following IMiD treatment in all three susceptible cell lines. An IMiD-mediated reduction of EZH2 and H3K27me3 levels was also observed in a multiple myeloma cell line. Furthermore, treatment with an EZH2-inhibitor reproduced the IMiD-mediated effects in HTLV-1-infected cells and multiple myeloma cells. These findings strongly suggest that a reduction of EZH2 expression is involved in the mechanism underlying the antitumor effects of IMiD.

Influence of the 5'-terminal sequences on the 5'-UTR structure of HIV-1 genomic RNA.

The 5'-UTR of HIV-1 genomic RNA is known to form specific structures and has important functions. There are three 5'-terminal sequences, G1, G2 and G3, with different localizations in the cell and virion particles as well as different efficiencies in translation and reverse transcription reactions. In the present study, the structural characteristics of the joint region between the TAR and PolyA stems was analysed, and it was found that small differences in the 5'-terminus affect the conformational characteristics of the stem-loop structures. In the G1 form, the two stems form a coaxial stem, whereas in the G2 and G3 forms, the two stems are structurally independent of each other. In the case of the G1 form, the 3'-flanking nucleotides of the PolyA stem are included in the stable coaxial stem structure, which may affect the rest of the 5'-UTR structure. This result demonstrates that the local conformation of this functionally key region has an important role in the function of the 5'-UTR.

Short-term cultured autologous peripheral blood mononuclear cells as a potential immunogen to activate Tax-specific CTL response in adult T-cell leukemia patients.

Activation of CD8+ Tax-specific CTL is a new therapeutic concept for adult T-cell leukemia (ATL) caused by HTLV-1. A recent clinical study of the dendritic cell vaccine pulsed with Tax peptides corresponding to CTL epitopes showed promising outcomes in ATL patients possessing limited human leukocyte antigen (HLA) alleles. In this study, we aimed to develop another immunotherapy to activate Tax-specific CTL without HLA limitation by using patients' own HTLV-1-infected cells as a vaccine. To examine the potential of HTLV-1-infected T-cells to activate CTL via antigen presenting cells, we established a unique co-culture system. We demonstrated that mitomycin C-treated HLA-A2-negative HTLV-1-infected T-cell lines or short-term cultured peripheral blood mononuclear cells (PBMC) derived from ATL patients induced cross-presentation of Tax antigen in co-cultured HLA-A2-positive antigen presenting cells, resulting in activation of HLA-A2-restricted CD8+ Tax-specific CTL. This effect was not inhibited by a reverse transcriptase inhibitor. IL-12 production and CD86 expression were also induced in antigen presenting cells co-cultured with HTLV-1-infected cells at various levels, which were improved by pre-treatment of the infected cells with histone deacetylase inhibitors. Furthermore, monocyte-derived dendritic cells induced from PBMC of a chronic ATL patient produced IL-12 and expressed enhanced levels of CD86 when co-cultured with autologous lymphocytes that had been isolated from the same PBMC and cultured for several days. These findings suggest that short-term cultured autologous PBMC from ATL patients could potentially serve as a vaccine to evoke Tax-specific CTL responses.

An Europium†(III) Luminophore with Pressure-Sensing Units: Effective Back Energy Transfer in Coordination Polymers with Hexadentate Porous Stable Networks.

A luminescent EuIII coordination polymer with O2 -sensing units under air, EuIII -hcpt (hcpt: 2,3,6,7,10,11-hexakis(4-carboxy-phenyl)triphenylene), is reported. The hexadentate carboxylic acids in hcpt ligands play an important role in the formation of tight-packed three-dimensional networks in EuIII -hcpt, giving hyper thermo-stable structures (decomposition temperature=420 °C). The three-dimensional porous network promotes bright luminescence (4f-4f emission quantum yield=70 %). The emission lifetime of EuIII -hcpt under vacuum (0.86 ms) was twice as large as that under O2 (0.48 ms, 1 atom:101.3 kPa). The Arrhenius analysis of the emission decay profile indicates that the back energy transfer (BEnT) from the emitting level of the europium(III) ion to the excited T1 state of the hcpt ligand should be activated at room temperature. The gradual decrease of emission lifetime is caused by the BEnT process in EuIII -hcpt. Finally, an advanced pressure-sensitive luminophore is demonstrated.

Near-IR Luminescent YbIII Coordination Polymers Composed of Pyrene Derivatives for Thermostable Oxygen Sensors.

Oxygen-sensitive and near-infrared (NIR) luminescent YbIII coordination polymers incorporating ligands based on pyrene derivatives were synthesized: YbIII -TBAPy and YbIII -TIAPy (TBAPy: 1,3,6,8-tetrakis(p-benzoate)pyrene; TIAPy: 1,3,6,8-tetrakis(3,5-isophthalic acid)pyrene). The coordination structures of these materials have been characterized by means of electrospray ionization mass spectrometry, X-ray diffraction analysis, and thermogravimetric analysis. Moreover, the porous structure of YbIII -TIAPy has been evaluated by measuring its N2 adsorption isotherm. The NIR luminescence properties of YbIII -TBAPy and YbIII -TIAPy have been examined by acquiring emission spectra and determining emission lifetimes under air or argon and in vacuo. YbIII -TIAPy exhibited high thermal stability (with a decomposition temperature of 400 °C), intense luminescence (with an emission quantum yield under argon of 6.6 %), and effective oxygen-sensing characteristics. These results suggest that NIR luminescent YbIII coordination polymers prepared using pyrene derivatives could have applications in novel thermo-stable oxygen sensors.

Impact of 5'-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription.

Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.

N-terminally truncated POM121C inhibits HIV-1 replication.

Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA) core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV) chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-ß/karyopherin subunit beta 1 (KPNB1). These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

IL-10-mediated signals act as a switch for lymphoproliferation in Human T-cell leukemia virus type-1 infection by activating the STAT3 and IRF4 pathways.

Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.

A novel mother-to-child human T-cell leukaemia virus type 1 (HTLV-1) transmission model for investigating the role of maternal anti-HTLV-1 antibodies using orally infected mother rats.

Human T-cell leukaemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukaemia/lymphoma (ATL) and is mainly transmitted from an infected mother to her child via breastfeeding. Such an HTLV-1 infection during childhood is believed to be a risk factor for ATL development. Although it has been suggested that an increased proviral load (PVL), a higher titre of antibody (Ab) in the infected mother and prolonged breastfeeding are associated with an increased risk of mother-to-child transmission (MTCT), the mechanisms underlying MTCT of HTLV-1 remain largely unknown. In this study, we developed an MTCT model using orally HTLV-1-infected rats that have no Ab responses against viral antigens, such as Gag and Env. In this model, HTLV-1 could be transmitted from the infected mother rats to their offspring at a high rate (50-100 %), and the rate of MTCT tended to be correlated with the PVL of the infected mother rats. Furthermore, passive immunization of uninfected adult rats and an infected mother rat with a rat anti-HTLV-1 Env gp46-neutralizing mAb was unable to suppress primary oral HTLV-1 infection to the adult rats and vertical HTLV-1 transmission to the offspring, respectively. Our findings indicate that this MTCT model would be useful to investigate not only the mechanisms of MTCT but also the role of anti-HTLV-1 Ab in MTCT of HTLV-1. They also provide some information on the role of maternal Abs in MTCT, which should be considered when designing a strategy for prevention of MTCT of HTLV-1.

HTLV-1 Tax-Specific CTL Epitope-Pulsed Dendritic Cell Therapy Reduces Proviral Load in Infected Rats with Immune Tolerance against Tax.

Adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8+ T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8+ T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE: Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.

Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels.

UNLABELLED: We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. IMPORTANCE: While human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving forces behind mutual evolution. The interplay of cellular APOBEC3G and viral Vif proteins is a typical example. Here, we demonstrate that naturally occurring single-nucleotide variations in the proximal region of splicing acceptor 1 (SA1prox) of the HIV-1 genome frequently alter Vif expression levels, thereby modulating viral replication potential in cells with various ABOBEC3G levels. The results of the present study reveal a previously unidentified and important way for HIV-1 to compete with APOBEC3G restriction by regulating its Vif expression levels. We propose that SA1prox plays a regulatory role in Vif counteraction against APOBEC3G in order to contribute to HIV-1 replication and evolution, and this may be applicable to other primate lentiviruses.

Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.

Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5'-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5'-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5'-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5'-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5'-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.

Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability.

UNLABELLED: We previously showed that prototype macaque-tropic human immunodeficiency virus type 1 (HIV-1) acquired nonsynonymous growth-enhancing mutations within a narrow genomic region during the adaptation process in macaque cells. These adaptive mutations were clustered in the 3' region of the pol gene, encoding a small portion of the C-terminal domain of integrase (IN). Mutations in HIV-1 IN have been reported to have pleiotropic effects on both the early and late phases in viral replication. cis-acting functions in the IN-coding sequence for viral gene expression have also been reported. We here demonstrated that the adaptive mutations promoted viral growth by increasing virion production with no positive effects on the early replication phase. Synonymous codon alterations in one of the adaptive mutations influenced virion production levels, which suggested nucleotide-dependent regulation. Indeed, when the single-nucleotide natural polymorphisms observed in the 3' regions of 196 HIV-1/simian immunodeficiency virus (SIVcpz) pol genes (nucleotides [nt] 4895 to 4929 for HIV-1 NL4-3) were introduced into macaque- and human-tropic HIV-1 clones, more than half exhibited altered replication potentials. Moreover, single-nucleotide mutations caused parallel increases or decreases in the expression levels of viral late proteins and viral replication potentials. We also showed that the overall expression profiles of viral mRNAs were markedly changed by single-nucleotide mutations. These results demonstrate that the 3' region of the HIV-1 pol gene (nt 4895 to 4929) can alter viral replication potential by modulating the expression pattern of viral mRNAs in a nucleotide-dependent manner. IMPORTANCE: Viruses have the plasticity to adapt themselves under various constraints. HIV-1 can mutate and evolve in growth-restrictive cells by acquiring adaptive changes in its genome. We have previously identified some growth-enhancing mutations in a narrow region of the IN-coding sequence, in which a number of cis-acting elements are located. We now focus on the virological significance of this pol gene region and the mechanistic basis underlying its effects on viral replication. We have found several naturally occurring synonymous mutations within this region that alter viral replication potentials. The effects caused by these natural single-nucleotide polymorphisms are linked to the definite expression patterns of viral mRNAs. We show here that the nucleotide sequence of the pol gene (nucleotides 4895 to 4929 for HIV-1 NL4-3) plays an important role in HIV-1 replication by modulating viral gene expression.

Interferon-α (IFN-α) suppresses HTLV-1 gene expression and cell cycling, while IFN-α combined with zidovudine induces p53 signaling and apoptosis in HTLV-1-infected cells.

BACKGROUND: Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs. RESULTS: ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs. CONCLUSIONS: IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL.