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Backgroud: Rett syndrome is a neurodevelopmental disorder that affects 1 in 10,000 females. Various treatments have been explored; however, no effective treatments have been reported to date, except for trofinetide, a synthetic analog of glycine-proline-glutamic acid, which was approved by the FDA in 2023. Serological biomarkers that correlate with the disease status of RTT are needed to promote early diagnosis and to develop novel agents. Methods: In this study, we performed a high-depth proteomic analysis of extracellular vesicles containing preparations extracted from patient plasma samples to identify novel biomarkers. Results: We identified 33 upregulated and 17 downregulated candidate proteins among a total of 4273 proteins in RTT compared to the healthy controls. Among these, UBE3B was predominantly increased in patients with Rett syndrome and exhibited a strong correlation with the clinical severity score, indicating the severity of the disease. Conclusions: We demonstrated that the proteomics of high-depth extracellular vesicles containing preparations in rare diseases could be valuable in identifying new disease biomarkers and understanding their pathophysiology.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

Thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome is a rare condition with diverse clinical and pathological characteristics related to multi-organ damage. We report a case of TAFRO syndrome complicated by immune thrombocytopenia with prolonged fever and thrombocytopenia for several weeks. A 61-year-old man was transferred with sepsis caused by Enterococcus faecalis, and developed disseminated intravascular coagulation. Antibiotics treatment was initiated: however, low-grade fever and thrombocytopenia persisted despite the adequate antimicrobial treatment. Systemic edema, pleural effusion, and ascites had developed before hospitalization, and renal and liver function had deteriorated, resulting in progressive multi-organ damage. Prednisolone 40 mg/day was initiated based on the assumption of a condition in which excessive production of inflammatory cytokines would lead to systemic deterioration and fatal organ damage. Subsequently, the fever resolved, and renal function began to normalize. However, thrombocytopenia did not show much recovery trend after Helicobacter pylori eradication therapy and initiation of thrombopoietin receptor agonists. Bone marrow biopsy results showed normal bone marrow with no malignant findings. Alternatively, significant clinical signs met the diagnostic criteria for TAFRO syndrome, and a renal biopsy revealed thrombotic microangiopathy, which is also reasonable for renal involvement in TAFRO syndrome. The use of cyclosporine remarkably corrected the thrombocytopenia. We considered this a case of TAFRO syndrome that developed after sepsis with disseminated intravascular coagulation and performed the differential diagnosis of prolonged thrombocytopenia and excluded it. Although TAFRO syndrome is a unique disease concept, diagnostic criteria may consist of nonspecific elements such as generalized edema, thrombocytopenia, persistent fever, and elevated inflammatory response, and there are many differential conditions to exclude, requiring caution in diagnosing TAFRO syndrome.

ImportanceHome monitoring of blood pressure (BP) in hypertensive patients can improve outcomes, but challenges to both patient compliance and the effective transmission of home BP readings to physicians can limit the extent to which physicians can use this information to improve care. The OMRON Hypertension Management Platform (OMRON HMP) pairs a home BP cuff with a digital product that tracks data, provides reminders to improve patient compliance, and provides a streamlined source of information to physicians.ObjectiveThe primary objective of the quality improvement (QI) project was to test the hypothesis that use of the OMRON HMP could reduce the number and cost of hypertension related claims, relative to a retrospectively matched cohort of insured members. A secondary objective was to demonstrate improvement in control of BP among patients.DesignEligible members were recruited to the QI project between December 1, 2018 and December 30, 2020 and data collected for six months following recruitment. All members received the OMRON HMP intervention.SettingEnrollment and data collection were coordinated on-site at selected PCP partner providers in Western Pennsylvania. Eligible members were identified from insurance claims data as those receiving care for primary hypertension from participating primary care physicians and/or cardiologists.ParticipantsEligible members were between the ages of 35 and 85, with a diagnosis of primary hypertension. The retrospective cohort was selected from electronic medical records of Highmark-insured patients with hypertension who received care at Allegheny Health Network (AHN), a subsidiary of Highmark Health. Members were matched on baseline BP and lipid measures, age, smoking status, diabetes status, race and sex.InterventionDaily home BP readings were recorded by the OMRON HMP app. Patient data was reviewed by clinical staff on a weekly basis and treatment plans could be adjusted in response to this data.ResultsOMRON HMP users showed a significant increase in the number and cost of hypertension-related claims, contrary to the hypothesis, but did display improvements in control of BP.Conclusions and RelevanceThe use of a digital platform to facilitate at-home BP monitoring appeared to improve BP control but led to increased hypertension-related costs in the short-term.

We investigated the applicability of ionic-liquid electrolytes to FeSi2/Si composite electrode for lithium-ion batteries. In conventional organic-liquid electrolytes, a discharge capacity of the electrode rapidly faded. In contrast, the electrode exhibited a superior cycle life with a reversible capacity of 1000 mA h g(Si)−1 over 850 cycles in a certain ionic-liquid electrolyte. The difference in the cycle life was explained by surface film properties. In addition, the rate performance of the FeSi2/Si electrode improved in another ionic-liquid electrolyte. Remarkably, lithiation of only Si in FeSi2/Si composite electrode occurred whereas each FeSi2- and Si-alone electrode alloyed with Li in the ionic-liquid electrolyte. FeSi2 certainly covered the shortcomings of Si and the FeSi2/Si composite electrode exhibited improved cycle life and rate capability compared to Si-alone electrode.

BackgroundSarcoidosis is a granulomatous systemic disease of unknown etiology. Mononuclear cells such as macrophages or lymphocytes in lung tissue and hilar or mediastinal lymph nodes have been recognized to play an essential role in granuloma formation in pulmonary sarcoidosis. Peripheral blood mononuclear cells (PBMCs) consist of several immunocompetent cells and have been shown to play a mechanistic role in the pathogenesis of sarcoidosis. However, the genetic modifications that occur in bulk PBMCs of sarcoidosis remain to be elucidated.PurposeThis study aimed to explore the pathobiological markers of sarcoidosis in PBMCs by comparing the transcriptional signature of PBMCs from patients with pulmonary sarcoidosis with those of healthy controls by RNA sequencing.MethodsPBMC samples were collected from subjects with pulmonary sarcoidosis with no steroid/immunosuppressant drugs (n = 8) and healthy controls (n = 11) from August 2020 to April 2021, and RNA sequencing was performed with the PBMC samples.ResultsPrincipal component analysis using RNA sequencing datasets comparing pulmonary sarcoidosis with healthy controls revealed that the two groups appeared to be differentiated, in which 270 differentially expressed genes were found in PBMCs between sarcoidosis and healthy controls. Enrichment analysis for gene ontology suggested that some biological processes related to the pathobiology of sarcoidosis, such as cellular response to interleukin (IL)-1 and IFN-γ, regulation of IL-6 production, IL-8 secretion, regulation of mononuclear cell migration, and response to lipopolysaccharide, were involved. Enrichment analysis of the KEGG pathway indicated the involvement of tumor necrosis factor (TNF), toll-like receptor signaling, IL-17 signaling pathways, phagosomes, and ribosomes. Most of the genes involved in TNF and IL-17 signaling pathways and phagosomes were upregulated, while most of the ribosome-related genes were downregulated.ConclusionThe present study demonstrated that bulk gene expression patterns in PBMCs were different between patients with pulmonary sarcoidosis and healthy controls. The changes in the gene expression pattern of PBMCs could reflect the existence of sarcoidosis lesions and influence granuloma formation in sarcoidosis. These new findings are important to strengthen our understanding of the etiology and pathobiology of sarcoidosis and indicate a potential therapeutic target for sarcoidosis.

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.

The accessory lentiviral protein X (Vpx) of the SIVsmm/mac and HIV-2 lineage targets the host-restriction factor SAMHD1 for proteasomal degradation. Here, the authors show that host PIM kinase-mediated phosphorylation of Vpx stabilizes its interaction with SAMHD1, suggesting PIM as potential antiviral targets.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.

Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.

Excessive inflammation in the lung is a primary cause of acute respiratory distress syndrome (ARDS). CD26/dipeptidyl peptidase-4 (DPP4) is a transmembrane protein that is expressed in various cell types and exerts multiple pleiotropic effects. We recently reported that pharmacological CD26/DPP4 inhibition ameliorated lipopolysaccharide (LPS)-induced lung injury in mice and exerted anti-inflammatory effects on human lung microvascular endothelial cells (HLMVECs), in vitro. However, the mechanistic roles of CD26/DPP4 in lung injury and its effects on HLMVECs remain unclear. In this study, transcriptome analysis, followed by various confirmation experiments using siRNA in cultured HLMVECs, are performed to evaluate the role of CD26/DPP4 in response to the pro-inflammatory involved in inflammation, barrier function, and regenerative processes in HLMVECs after pro-inflammatory stimulation. These are all functions that are closely related to the pathophysiology and repair process of lung injury. Confirmatory experiments using flow cytometry; enzyme-linked immunosorbent assay; quantitative polymerase chain reaction; dextran permeability assay; WST-8 assay; wound healing assay; and tube formation assay, reveal that the reduction of CD26/DPP4 via siRNA is associated with altered parameters of inflammation, barrier function, and the regenerative processes in HLMVECs. Thus, CD26/DPP4 can play a pathological role in mediating injury in pulmonary endothelial cells. CD26/DPP4 inhibition can be a new therapeutic strategy for inflammatory lung diseases, involving pulmonary vascular damage.

The stalk domain of influenza virus envelope glycoprotein hemagglutinin (HA) constitutes the axis connecting the head and transmembrane domains, and plays pivotal roles in conformational rearrangements of HA for virus infection. Here we characterized molecular interactions between the anti-HA stalk neutralization antibody F11 and influenza A(H1N1)pdm09 HA to understand the structural basis of the actions and modifications of this antibody. In silico structural analyses using a model of the trimeric HA ectodomain indicated that the F11 Fab fragment has physicochemical properties, allowing it to crosslink two HA monomers by binding to a region near the proteolytic cleavage site of the stalk domain. Interestingly, the F11 binding allosterically caused a marked suppression of the structural dynamics of the HA cleavage loop and flanking regions. Structure-guided mutagenesis of the F11 antibody revealed a critical residue in the F11 light chain for the F11-mediated neutralization. Finally, the mutagenesis led to identification of a unique F11 derivative that can neutralize both F11-sensitive and F11-resistant A(H1N1)pdm09 viruses. These results raise the possibility that F11 sterically and physically disturbs proteolytic cleavage of HA for the ordered conformational rearrangements and suggest that in silico guiding experiments can be useful to create anti-HA stalk antibodies with new phenotypes.

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1NL4-3 adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.

The capsid of human immunodeficiency virus type 1 (HIV-1) is a shell that encloses viral RNA and is highly conserved among many strains of the virus. It forms a conical structure by assembling oligomers of capsid (CA) proteins. CA dysfunction is expected to be an important target of suppression of HIV-1 replication, and it is important to understand a new mechanism that could lead to the CA dysfunction. A drug targeting CA however, has not been developed to date. Hydrophobic interactions between two CA molecules via Trp184/Met185 in CA were recently reported to be important for stabilization of the multimeric structure of CA. In the present study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site, was synthesized and its significant anti-HIV-1 activity was confirmed. Structure activity relationship (SAR) studies of its derivatives were performed and provided results that are expected to be useful in the future design and development of novel anti-HIV agents targeting CA.

We present a case of a 37-year-old female who complained of frequent palpitations caused by an atrial tachycardia and atrial premature contractions. Angiography revealed that the coronary sinus was occluded at the ostium and connected to a persistent left superior vena cava. An electrophysiological study and three-dimensional mapping revealed that the origin of the atrial tachycardia and atrial premature contractions was at the coronary sinus ostium in the right atrium. After repeat applications of radiofrequency energy at that site, no further atrial tachycardia or atrial premature contractions were induced by atrial burst pacing. To the best of our knowledge, this is the first report of an atrial tachycardia originating from an occluded coronary sinus ostium.

Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health.

Genomic RNA of HIV-1 contains localized structures critical for viral replication. Its structural analysis has demonstrated a stem-loop structure, SLSA1, in a nearby region of HIV-1 genomic splicing acceptor 1 (SA1). We have previously shown that the expression level of vif mRNA is considerably altered by some natural single-nucleotide variations (nSNVs) clustering in SLSA1 structure. In this study, besides eleven nSNVs previously identified by us, we totally found nine new nSNVs in the SLSA1-containing sequence from SA1, splicing donor 2, and through to the start codon of Vif that significantly affect the vif mRNA level, and designated the sequence SA1D2prox (142 nucleotides for HIV-1 NL4-3). We then examined by extensive variant and mutagenesis analyses how SA1D2prox sequence and SLSA1 secondary structure are related to vif mRNA level. While the secondary structure and stability of SLSA1 was largely changed by nSNVs and artificial mutations introduced to restore the original NL4-3 form from altered ones by nSNVs, no clear association of the two SLSA1 properties with vif mRNA level was observed. In contrast, when naturally occurring SA1D2prox sequences that contain multiple nSNVs were examined, we attained significant inverse correlation between the vif level and SLSA1 stability. These results may suggest that SA1D2prox sequence adapts over time, and also that the altered SA1D2prox sequence, SLSA1 stability, and vif level are mutually related. In total, we show here that the entire SA1D2prox sequence and SLSA1 stability critically contribute to the modulation of vif mRNA level.

CD8+ T-cell responses exert strong suppressive pressure on HIV replication and select for viral escape mutations. Some of these major histocompatibility complex class I (MHC-I)-associated mutations result in reduction of in vitro viral replicative capacity. While these mutations can revert after viral transmission to MHC-I-disparate hosts, recent studies have suggested that these MHC-I-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Here, we directly show an increase in the in vivo virulence of an MHC-I-adapted virus serially-passaged through MHC-I-mismatched hosts in a macaque AIDS model despite a reduction in in vitro viral fitness. The first passage simian immunodeficiency virus (1pSIV) obtained 1 year after SIVmac239 infection in a macaque possessing a protective MHC-I haplotype 90-120-Ia was transmitted into 90-120-Ia- macaques, whose plasma 1 year post-infection was transmitted into other 90-120-Ia- macaques to obtain the third passage SIV (3pSIV). Most of the 90-120-Ia-associated mutations selected in 1pSIV did not revert even in 3pSIV. 3pSIV showed lower in vitro viral fitness but induced persistent viremia in 90-120-Ia- macaques. Remarkably, 3pSIV infection in 90-120-Ia+ macaques resulted in significantly higher viral loads and reduced survival compared to wild-type SIVmac239. These results indicate that MHC-I-adapted SIVs serially-transmitted through MHC-I-mismatched hosts can have higher virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that multiply-passaged HIVs could result in loss of HIV-specific CD8+ T cell responses in human populations and the in vivo pathogenic potential of these escaped viruses may be enhanced.

Feline calicivirus (FCV) protease functions to cleave viral precursor proteins during productive infection. Previous studies have mapped a protease-coding region and six cleavage sites in viral precursor proteins. However, how the FCV protease interacts with its substrates remains unknown. To gain insights into the interactions, we constructed a molecular model of the FCV protease bound with the octapeptide containing a cleavage site of the capsid precursor protein by homology modeling and docking simulation. The complex model was used to screen for the substrate mimic from a chemical library by pharmacophore-based in silico screening. With this structure-based approach, we identified a compound that has physicochemical features and arrangement of the P3 and P4 sites of the substrate in the protease, is predicted to bind to FCV proteases in a mode similar to that of the authentic substrate, and has the ability to inhibit viral protease activity in vitro and in the cells, and to suppress viral replication in FCV-infected cells. The complex model was further subjected to molecular dynamics simulation to refine the enzyme-substrate interactions in solution. The simulation along with a variation study predicted that the authentic substrate and anti-FCV compound share a highly conserved binding site. These results suggest the validity of our in silico model for elucidating protease-substrate interactions during FCV replication and for developing antivirals.

Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A-H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5) derived from HIV-1(JR-FLan) is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan) to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines.

Human immunodeficiency virus (HIV) type 2 shows limited geographical distribution compared with HIV type 1. Although 8 genetic groups of HIV type 2 (HIV-2) have been described, recombinant viruses between these groups are rarely observed. Recently, three HIV-2 patients in Japan were described with rapidly progressive, acquired immunodeficiency. These patients were infected with an A/B inter-group recombinant designated CRF01_AB. Here, we characterize the capsid protein (CA) encoded by the viruses from these patients. HIV-2 CRF01_AB CA showed unique amino acid sequence almost equally distinct from group A and group B viruses. Notably, HIV-2 CRF01_AB CA showed potent resistance to human TRIM5α. In addition to the previously identified amino acid position 119 in the N-terminal domain of CA, we found that HIV-2 CRF01_AB-specific amino acid substitutions in the C-terminal domain also were necessary for resistance to human TRIM5α. These results indicate that retroviruses can evade TRIM5α by substitution at residues within the C-terminal domain of CA.

BACKGROUND: The net charge of the hypervariable V3 loop on the HIV-1 envelope gp120 outer domain plays a key role in modulating viral phenotype. However, the molecular mechanisms underlying the modulation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: By combining computational and experimental approaches, we examined how V3 net charge could influence the phenotype of the gp120 interaction surface. Molecular dynamics simulations of the identical gp120 outer domain, carrying a V3 loop with net charge of +3 or +7, showed that the V3 change alone could induce global changes in fluctuation and conformation of the loops involved in binding to CD4, coreceptor and antibodies. A neutralization study using the V3 recombinant HIV-1 infectious clones showed that the virus carrying the gp120 with +3 V3, but not with +7 V3, was resistant to neutralization by anti-CD4 binding site monoclonal antibodies. An information entropy study shows that otherwise variable surface of the gp120 outer domain, such as V3 and a region around the CD4 binding loop, are less heterogeneous in the gp120 subpopulation with +3 V3. CONCLUSIONS/SIGNIFICANCE: These results suggest that the HIV-1 gp120 V3 loop acts as an electrostatic modulator that influences the global structure and diversity of the interaction surface of the gp120 outer domain. Our findings will provide a novel structural basis to understand how HIV-1 adjusts relative replication fitness by V3 mutations.

Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.

We previously reported that human immunodeficiency virus type 2 (HIV-2) carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA) could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM). To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7) affected conformation of the neighboring loop between helices 4 and 5 (L4/5), and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A) of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5α recognition.

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

BACKGROUND: Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a DNA polymerase that converts viral RNA genomes into proviral DNAs. How HIV-1 RT regulates nucleotide selectivity is a central issue for genetics and the nucleoside analog RT inhibitor (NRTI) resistance of HIV-1. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that an ATP molecule at physiological concentrations acts as an allosteric regulator of HIV-1 RT to decrease the K(m) value of the substrate, decrease the k(cat) value, and increase the K(i) value of NRTIs for RT. Computer-assisted structural analyses and mutagenesis studies suggested the positions of the ATP molecule and NRTI-resistance mutations during a catalytic reaction, which immediately predict possible influences on nucleotide insertion into the catalytic site, the DNA polymerization, and the excision reaction. CONCLUSIONS/SIGNIFICANCE: These data imply that the ATP molecule and NRTI mutations can modulate nucleotide selectivity by altering the fidelity of the geometric selection of nucleotides and the probability of an excision reaction.

The third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism and host humoral immune responses. Positive charge of the V3 plays a key role in determining viral coreceptor tropism. Here, we examined by bioinformatics, experimental, and protein modelling approaches whether the net positive charge of V3 sequence regulates viral sensitivity to humoral immunity. We chose HIV-1 CRF01_AE strain as a model virus to address the question. Diversity analyses using CRF01_AE V3 sequences from 37 countries during 1984 and 2005 (n = 1361) revealed that reduction in the V3's net positive charge makes V3 less variable due to limited positive selection. Consistently, neutralization assay using CRF01_AE V3 recombinant viruses (n = 30) showed that the reduction in the V3's net positive charge rendered HIV-1 less sensitive to neutralization by the blood anti-V3 antibodies. The especially neutralization resistant V3 sequences were the particular subset of the CCR5-tropic V3 sequences with net positive charges of +2 to +4. Molecular dynamics simulation of the gp120 monomers showed that the V3's net positive charge regulates the V3 configuration. This and reported gp120 structural data predict a less-exposed V3 with a reduced net positive charge in the native gp120 trimer context. Taken together, these data suggest a key role of the V3's net positive charge in the immunological escape and coreceptor tropism evolution of HIV-1 CRF01_AE in vivo. The findings have molecular implications for the adaptive evolution and vaccine design of HIV-1.