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ABSTRACT Neuraminidase (NA) is a pivotal surface enzyme and a key therapeutic target in combating the influenza A virus. Its evolution can lead to potential zoonotic transmission, seasonal epidemics, and the emergence of drug-resistant mutants. To gain comprehensive insights into the mutational effects and drug resistance profiles of NA, we employed a high-throughput profiling system to quantify the replication capacity of NA mutants at the single-nucleotide level in mouse lung tissues. The fitness of NA mutants is generally correlated with natural mutation occurrence and is constrained by both the requirement to maintain protein stability and NA function. Leveraging this system, we profiled the drug resistance to the three most commonly used neuraminidase inhibitors (NAIs): zanamivir, oseltamivir, and peramivir. In addition to identifying previously reported drug resistance mutations, we validated novel mutants. Notably, we identified an allosteric mutation that confers resistance to all three drugs, which may affect drug binding by interfering with the tetramerization of NA. Moreover, the fitness cost associated with drug-resistant mutations may limit their widespread dissemination. In summary, we provided a parallel characterization of NA’s fitness and drug resistance landscape in an in vivo context, which may guide the rational selection of antiviral drugs for optimal therapeutic efficacy and second-generation NAI development. IMPORTANCE NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA’s mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

Here, Kim et al. describe a new kick and kill strategy utilizing a single administration of a protein kinase C modulator and latency reversing agent in combination with injections of allogeneic peripheral blood natural killer cells diminishes the HIV reservoir in HIV-infected humanized mice.

In this paper, we propose a method to evaluate the motor coordination of runners based on the analysis of amplitude and spatiotemporal dynamics of multichannel electromyography. A new diagnostic index for the coordination of runners was proposed, including the amplitude of electromyography, the spatiotemporal stability coefficient, and the symmetry coefficient of muscle force. The motor coordination of 13 professional runners was studied. Detailed anthropometric information was recorded about the professional runners. It has been found that professional athletes are characterized by the stability of movement repetition (more than 83%) and the high degree of symmetry of muscle efforts of the left and right legs (more than 81%) regardless of the changes in load during running at a speed of 8–12 km/hr. Scientific and technological means can support the scientific training of athletes. The end of the Winter Olympic Games has shown us the powerful power of a series of intelligent scientific equipment, including electro-magnetic gun, in sports training. We also look forward to the continuous innovation of these advanced technologies, which will contribute to the intelligent development of sports scientific research.

Bone cancer pain (BCP) is excruciating for cancer patients, with limited clinical treatment options and significant side effects, due to the complex and unclear pathogenesis of bone cancer pain. Peripheral sensitization in dorsal root ganglion (DRG) neurons is a recognized cellular mechanism for bone cancer pain. The pathological mechanism of chronic pain is increasingly being affected by epigenetic mechanisms. In this study, we unbiasedly showed that the DNA hydroxymethylase ten-eleven translocation 1 (TET1) expression was significantly increased in the L4–6 DRG of BCP rats and ten-eleven translocation 2 (TET2) expression did not change significantly. Notably, TET1 inhibition by intrathecal injection of Bobcat339 (a TET1 inhibitor) effectively relieved mechanical hyperalgesia in BCP rats. Peripheral sensitization in chronic pain relies on the activation and overexpression of ion channels on neurons. Here, we demonstrated that TRPV4, one of the transient receptor potential ion channel family members, was significantly elevated in the L4–6 DRG of BCP rats. In addition, TRPV4 inhibition by intrathecal injection of HC067047 (a TRPV4 inhibitor) also significantly attenuated mechanical hyperalgesia in BCP rats. Interestingly, we found that TET1 inhibition downregulated TRPV4 expression in the L4–6 DRG of BCP rats. As a result, these findings suggested that TET1 may contribute to bone cancer pain by upregulating TRPV4 expression in the L4–6 DRG of BCP rats and that TET1 or TRPV4 may become therapeutic targets for bone cancer pain.

Identification of low abundance proteins interacting with viral proteins can be challenging. Here, Du et al. develop an mRNA display approach with a library of even distribution, identify host proteins interacting with NS1 protein of influenza A virus, and show that one interactor provides a means to regulate cellular fatty acids synthesis.

Abstract Background Deficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues. Results In addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways. Conclusion These results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders.

Abstract Background Recent advancements in next-generation sequencing have rapidly improved our ability to study genomic material at an unprecedented scale. Despite substantial improvements in sequencing technologies, errors present in the data still risk confounding downstream analysis and limiting the applicability of sequencing technologies in clinical tools. Computational error correction promises to eliminate sequencing errors, but the relative accuracy of error correction algorithms remains unknown. Results In this paper, we evaluate the ability of error correction algorithms to fix errors across different types of datasets that contain various levels of heterogeneity. We highlight the advantages and limitations of computational error correction techniques across different domains of biology, including immunogenomics and virology. To demonstrate the efficacy of our technique, we apply the UMI-based high-fidelity sequencing protocol to eliminate sequencing errors from both simulated data and the raw reads. We then perform a realistic evaluation of error-correction methods. Conclusions In terms of accuracy, we find that method performance varies substantially across different types of datasets with no single method performing best on all types of examined data. Finally, we also identify the techniques that offer a good balance between precision and sensitivity.

Rejection continues to be an important cause of graft loss in solid organ transplantation, but deep exploration of intragraft alloimmunity has been limited by the scarcity of clinical biopsy specimens. Emerging single cell immunoprofiling technologies have shown promise in discerning mechanisms of autoimmunity and cancer immunobiology. Within these applications, Imaging Mass Cytometry (IMC) has been shown to enable highly multiplexed, single cell analysis of immune phenotypes within fixed tissue specimens. In this study, an IMC panel of 10 validated markers was developed to explore the feasibility of IMC in characterizing the immune landscape of chronic rejection (CR) in clinical tissue samples obtained from liver transplant recipients. IMC staining was highly specific and comparable to traditional immunohistochemistry. A single cell segmentation analysis pipeline was developed that enabled detailed visualization and quantification of 109,245 discrete cells, including 30,646 immune cells. Dimensionality reduction identified 11 unique immune subpopulations in CR specimens. Most immune subpopulations were increased and spatially related in CR, including two populations of CD45+/CD3+/CD8+ cytotoxic T-cells and a discrete CD68+ macrophage population, which were not observed in liver with no rejection (NR). Modeling via principal component analysis and logistic regression revealed that single cell data can be utilized to construct statistical models with high consistency (Wilcoxon Rank Sum test, p=0.000036). This study highlights the power of IMC to investigate the alloimmune microenvironment at a single cell resolution during clinical rejection episodes. Further validation of IMC has the potential to detect new biomarkers, identify therapeutic targets, and generate patient-specific predictive models of clinical outcomes in solid organ transplantation.

It has been reported that multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) including Alpha, Beta, Gamma, and Delta can reduce neutralization by antibodies, resulting in vaccine breakthrough infections. Virus–antiserum neutralization assays are typically performed to monitor potential vaccine breakthrough strains. However, experiment-based methods took several weeks whether newly emerging variants can break through current vaccines or therapeutic antibodies. To address this, we sought to establish a computational model to predict the antigenicity of SARS-CoV-2 variants by sequence alone. In this study, we firstly identified the relationship between the antigenic difference transformed from the amino acid sequence and the antigenic distance from the neutralization titers. Based on this correlation, we obtained a computational model for the receptor-binding domain (RBD) of the spike protein to predict the fold decrease in virus–antiserum neutralization titers with high accuracy (~0.79). Our predicted results were comparable to experimental neutralization titers of variants, including Alpha, Beta, Delta, Gamma, Epsilon, Iota, Kappa, and Lambda, as well as SARS-CoV. Here, we predicted the fold of decrease of Omicron as 17.4-fold less susceptible to neutralization. We visualized all 1,521 SARS-CoV-2 lineages to indicate variants including Mu, B.1.630, B.1.633, B.1.649, and C.1.2, which can induce vaccine breakthrough infections in addition to reported VOCs Beta, Gamma, Delta, and Omicron. Our study offers a quick approach to predict the antigenicity of SARS-CoV-2 variants as soon as they emerge. Furthermore, this approach can facilitate future vaccine updates to cover all major variants. An online version can be accessed at http://jdlab.online.

Today, more and more Internet public media platforms allowing people to make donations or seek help are being founded in China. However, there are few specialized sports-related public welfare platforms. In this paper, a sports-related public welfare platform that aims to help people who were disabled due to participation in sports and those who are disabled but want to participate in sports was developed based on multi-sensor technology. A multi-sensor data fusion algorithm was developed, and its estimation performance was verified by comparing it with the existing Kalman consistent filtering algorithm in terms of average estimation and average consistency errors. Experimental results prove that the speed of the data collection and analysis of the sports-related public welfare platform using the algorithm established in this paper was greatly improved. Relevant data on how users used this platform showed that various factors affected users’ practical satisfaction with sports-related public welfare media platforms. It is suggested that a sports-related public welfare media platform should pay attention to the aid effect, and specific efforts should be devoted to improving the reliability and timeliness of public welfare aid information, and ensuring the stability of the platform system.

Abstract Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS‐CoV‐2 Spike protein binding to the human angiotensin‐converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS‐CoV‐2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient‐specific‐vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS‐CoV‐2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome‐hACE2 and anti‐Interleukin (IL) 6 antibodies.

ABSTRACT RNA viruses, such as hepatitis C virus (HCV), influenza virus, and SARS-CoV-2, are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes, especially under different selection conditions. Here, we systematically quantified the distribution of fitness effects of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of HCV. We found that the majority of nonsynonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized. The replication fitness of viruses is correlated with the pattern of sequence conservation in nature, and viral evolution is constrained by the need to maintain protein stability. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug daclatasvir at multiple concentrations. Both the relative fitness values and the number of beneficial mutations were found to increase with the increasing concentrations of daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. Overall, our results show that the distribution of fitness effects of mutations is modulated by both the constraints on the biophysical properties of proteins (i.e., selection pressure for protein stability) and the level of environmental stress (i.e., selection pressure for drug resistance). IMPORTANCE Many viruses adapt rapidly to novel selection pressures, such as antiviral drugs. Understanding how pathogens evolve under drug selection is critical for the success of antiviral therapy against human pathogens. By combining deep sequencing with selection experiments in cell culture, we have quantified the distribution of fitness effects of mutations in hepatitis C virus (HCV) NS5A protein. Our results indicate that the majority of single amino acid substitutions in NS5A protein incur large fitness costs. Simulation of protein stability suggests viral evolution is constrained by the need to maintain protein stability. By subjecting the mutant viruses to selection under an antiviral drug, we find that the adaptive potential of viral proteins in a novel environment is modulated by the level of environmental stress, which can be explained by a pharmacodynamics model. Our comprehensive characterization of the fitness landscapes of NS5A can potentially guide the design of effective strategies to limit viral evolution.

Drug-resistant mutations often have deleterious impacts on replication fitness, posing a fitness cost that can only be overcome by compensatory mutations. However, the role of fitness cost in the evolution of drug resistance has often been overlooked in clinical studies or in vitro selection experiments, as these observations only capture the outcome of drug selection. In this study, we systematically profile the fitness landscape of resistance-associated sites in HIV-1 protease using deep mutational scanning. We construct a mutant library covering combinations of mutations at 11 sites in HIV-1 protease, all of which are associated with resistance to protease inhibitors in clinic. Using deep sequencing, we quantify the fitness of thousands of HIV-1 protease mutants after multiple cycles of replication in human T cells. Although the majority of resistance-associated mutations have deleterious effects on viral replication, we find that epistasis among resistance-associated mutations is predominantly positive. Furthermore, our fitness data are consistent with genetic interactions inferred directly from HIV sequence data of patients. Fitness valleys formed by strong positive epistasis reduce the likelihood of reversal of drug resistance mutations. Overall, our results support the view that strong compensatory effects are involved in the emergence of clinically observed resistance mutations and provide insights to understanding fitness barriers in the evolution and reversion of drug resistance.

Summary: Zika virus (ZIKV) infection causes Guillain-Barré syndrome and severe birth defects. ZIKV envelope (E) protein is the major viral protein involved in cell receptor binding and entry and is therefore considered one of the major determinants in ZIKV pathogenesis. Here we report a gene-wide mapping of functional residues of ZIKV E protein using a mutant library, with changes covering every nucleotide position. By comparing the replication fitness of every viral mutant between mosquito and human cells, we identified that mutations affecting glycosylation display the most divergence. By characterizing individual mutants, we show that ablation of glycosylation selectively benefits ZIKV infection of mosquito cells by enhancing cell entry, whereas it either has little impact on ZIKV infection on certain human cells or leads to decreased infection through the entry factor DC-SIGN. In conclusion, we define the roles of individual residues of ZIKV envelope protein, which contribute to ZIKV replication fitness in human and mosquito cells. : Entomology; Genomics; Virology; Protein Structure Aspects Subject Areas: Entomology, Genomics, Virology, Protein Structure Aspects

Database systems have a large number of configuration parameters that control functional and non-functional properties (e.g., performance and cost). Different configurations may lead to different performance values. To understand and predict the effect of configuration parameters on system performance, several learning-based strategies have been recently proposed. However, existing approaches usually assume a fixed database version such that learning has to be repeated once the database version changes. Repeating measurement and learning for each version is expensive and often practically infeasible. Instead, we propose the Partitioned Co-Kriging (PCK) approach that transfers knowledge from an older database version (source domain) to learn a reliable performance prediction model fast for a newer database version (target domain). Our method is based on the key observations that performance responses typically exhibit similarities across different database versions. We conducted extensive experiments under 5 different database systems with different versions to demonstrate the superiority of PCK. Experimental results show that PCK outperforms six state-of-the-art baseline algorithms in terms of prediction accuracy and measurement effort.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions.

Summary: The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions. : Cerebral organoids recapitulate many aspects of human corticogenesis and are a useful platform for modeling neurodevelopmental mechanisms and diseases. Watanabe et al. describe enhanced organoid methods and model ZIKV pathology. More susceptibility receptors for ZIKV are identified, and differential effects of various compounds to mitigate ZIKV-induced cytopathy are demonstrated. Keywords: neurogenesis, neural stem cell, embryonic stem cell, organoid, differentiation, neural development, cerebral cortex, Zika virus, human brain

As cancer is one of the major fatal diseases for human beings worldwide, the metastasis of tumor cells (TCs) from a blood vessel to an adjacent organ has become a focus of research. A tumor metastasis theory named the “two-step theory” pointed out that polymorphnuclear neutrophils (PMNs) could facilitate TC adhesion on an endothelial monolayer under flow, which was regulated by shear flow and promoted by fibrinogen and fibrin. In order to further understand the role of hydrodynamics played in the “two-step theory”, we improved our side-view micro-particle imaging velocimetry (PIV) system and successfully measured the flow velocity profiles around adherent PMNs and TCs on an endothelial monolayer in the presence of soluble fibrinogen or fibrin under shear flow. Combined with a computational fluid dynamics simulation, we found that: (1) soluble fibrinogen and fibrin influenced the variations of relative shear rates above an adhered PMN and an adherent TC at different PMN-to-TC position states; (2) compared with soluble fibrinogen, soluble fibrin made the curves of relative shear rates above an adherent cell flatter. Soluble fibrin might increase the collision frequency and affect the contact time and contact area between PMNs, TCs, and endothelium cells, resulting in the enhancement of TC adhesion and retention on an endothelial monolayer.

Gamma interferon (IFN-γ) is known to negatively regulate murine gammaherpesvirus-68 (MHV-68 or γHV-68) replication. This process involves the suppression of the viral gene replication and transcription activator (RTA) promoter, as well as activation of signal transducers and activators of transcription (STAT1). Notably, this effect is gradually attenuated during MHV-68 infection of bone marrow-derived macrophages (BMMs), which raised the possibility that the virus may utilize a mechanism that counteracts the antiviral effect of IFN-γ. By identifying the cellular factors that negatively regulate JAK-STAT1 signaling, we revealed that the infection of BMMs by MHV-68 induces the expression of suppressor of cytokine signaling 1 (SOCS1) and that depletion of SOCS1 restores the inhibitory effect of IFN-γ on virus replication. Moreover, we demonstrated that the expression of SOCS1 was induced as a result of the Toll-like receptor 3 (TLR3) mediated activation of the NF-κB signaling cascade. In conclusion, we report that TLR3-TRAF-NF-κB signaling pathway play a role in the induction of SOCS1 that counteracts the antiviral effect of IFN-γ during MHV-68 infection. This process is cell type-specific: it is functional in macrophages, but not in epithelial cells or fibroblasts. Our study reveals a mechanism that balances the immune responses and the escape of a gamma-herpesvirus in some antigen-presenting cells.

ABSTRACT Certain “protective” major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8+ cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo. Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape.

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.

[This corrects the article DOI: 10.1371/journal.pgen.1005310.].

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.

We report experimental investigation of the photoluminescence (PL) generated from the gold nanoshells of the dielectric-metal core-shell resonators (DMCSR) that support multipolar electric and magnetic based cavity plasmon resonances. Significantly enhanced and modulated PL spectrum is observed. By comparing the experimental results with analytical Mie calculations, we are able to demonstrate that the observed reshaping effects are due to the excitations of those narrow-band cavity plasmon resonances. We also present that the variation on the dielectric core size allows for tuning the cavity plasmon resonance wavelengths and thus the peak positions of the PL spectrum.

The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 204 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve.

Murine herpesvirus-68 (MHV-68) productively infects mouse lungs, exhibiting a complex pathology characteristic of both acute viral infections and chronic respiratory diseases. We sought to discover proteins differentially expressed in bronchoalveolar lavage (BAL) from mice infected with MHV-68. Mice were infected intranasally with MHV-68. After nine days, as the lytic phase of infection resolved, differential BAL proteins were identified by two-dimensional (2D) electrophoresis and mass spectrometry. Of 23 unique proteins, acute phase proteins, vitamin A transport, and oxidative stress response factors Pdx6 and EC-SOD (Sod3) were enriched. Correspondingly, iNOS2 was induced in lung tissue by seven days post-infection. Oxidative stress was partly a direct result of MHV-68 infection, as reactive oxygen species (ROS) were induced in cultured murine NIH3T3 fibroblasts and human lung A549 cells infected with MHV-68. Finally, mice infected with a recombinant MHV-68 co-expressing inflammatory cytokine murine interleukin 6 (IL6) showed exacerbated oxidative stress and soluble type I collagen characteristic of tissue recovery. Thus, oxidative stress appears to be a salient feature of MHV-68 pathogenesis, in part caused by lytic replication of the virus and IL6. Proteins and small molecules in lung oxidative stress networks therefore may provide new therapeutic targets to ameliorate respiratory virus infections.

Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

Recent discoveries of direct acting antivirals against Hepatitis C virus (HCV) have raised hopes of effective treatment via combination therapies. Yet rapid evolution and high diversity of HCV populations, combined with the reality of suboptimal treatment adherence, make drug resistance a clinical and public health concern. We develop a general model incorporating viral dynamics and pharmacokinetics/ pharmacodynamics to assess how suboptimal adherence affects resistance development and clinical outcomes. We derive design principles and adaptive treatment strategies, identifying a high-risk period when missing doses is particularly risky for de novo resistance, and quantifying the number of additional doses needed to compensate when doses are missed. Using data from large-scale resistance assays, we demonstrate that the risk of resistance can be reduced substantially by applying these principles to a combination therapy of daclatasvir and asunaprevir. By providing a mechanistic framework to link patient characteristics to the risk of resistance, these findings show the potential of rational treatment design.

Recent advances in DNA sequencing capacity to accurately quantify the copy number of individual variants in a large and diverse population allows in parallel determination of the phenotypic effects caused by each genetic modification. This systematic profiling approach is a combination of forward and reverse genetics, which we refer to as quantitative high-resolution genetics (qHRG). This protocol describes how to determine the relative fitness score of each variant compared to wild type (WT) virus based on its frequency determined by Illumina sequencing. Random mutagenesis techniques will be used to introduce randomization at each codon position of the targeted region, thereby generating a comprehensive input mutant library with substitutions at each position of interest (Qi et al., 2014; Wu et al., 2014a; Wu et al., 2014b). After selection, each selected library will be sequenced by Illumina paired-end sequencing and the frequency of each mutation will be determined. Based on the change in frequency, the relative fitness score of each mutant can be calculated with regression analysis.

ABSTRACT Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains. IMPORTANCE Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.

Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼ 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼ 0.005% to ∼ 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.

Genome-wide yeast two-hybrid (Y2H) screens were conducted to elucidate the molecular functions of open reading frames (ORFs) encoded by murine γ-herpesvirus 68 (MHV-68). A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs). Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP) and low priority/not-scored (Y2H-LP/NS) groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR-/- mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68.

The ability to control cellular functions can bring about many developments in basic biological research and its applications. The presence of multiple signals, internal as well as externally imposed, introduces several challenges for controlling cellular functions. Additionally the lack of clear understanding of the cellular signaling network limits our ability to infer the responses to a number of signals. This work investigates the control of Kaposi's sarcoma-associated herpesvirus reactivation upon treatment with a combination of multiple signals. We utilize mathematical model-based as well as experiment-based approaches to achieve the desired goals of maximizing virus reactivation. The results show that appropriately selected control signals can induce virus lytic gene expression about ten folds higher than a single drug; these results were validated by comparing the results of the two approaches, and experimentally using multiple assays. Additionally, we have quantitatively analyzed potential interactions between the used combinations of drugs. Some of these interactions were consistent with existing literature, and new interactions emerged and warrant further studies. The work presents a general method that can be used to quantitatively and systematically study multi-signal induced responses. It enables optimization of combinations to achieve desired responses. It also allows identifying critical nodes mediating the multi-signal induced responses. The concept and the approach used in this work will be directly applicable to other diseases such as AIDS and cancer.

Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKbeta pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKbeta impaired the lytic replication of gamma-herpesvirus 68 (gammaHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. gammaHV68 infection activated IKKbeta in a MAVS-dependent manner; however, IKKbeta phosphorylated and promoted the transcriptional activation of the gammaHV68 replication and transcription activator (RTA). Mutational analyses identified IKKbeta phosphorylation sites, through which RTA-mediated transcription was increased by IKKbeta, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant gammaHV68 carrying mutations within the IKKbeta phosphorylation sites was greatly impaired. These findings support the conclusion that gammaHV68 hijacks the antiviral MAVS-IKKbeta pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation.

Gamma-herpesviruses (gammaHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine gamma-herpesvirus 68 (gammaHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant gammaHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which gammaHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of gamma-herpesviruses.

Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3' non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

Objective To evaluate the short-term efficacy and safety of two Janus kinase (JAK) 1 inhibitors, upadacitinib and abrocitinib, in the treatment of atopic dermatitis of the head and neck. Methods This study retrospectively analyzed the clinical data of patients with atopic dermatitis of the head and neck who received upadacitinib and abrocitinib in the Department of Dermatology, the First Affiliated Hospital of Soochow University from January 2022 to June 2023. At 0,2,4,8, and 12 weeks after treatment, clinical symptoms and itching of patients were scored using the eczema area and severity index (EASI),investigator's global assessment (IGA), visual analogue scale (VAS) itch score, and the body surface area (BSA), to evaluate the efficacy and safety of treatment. Meanwhile, the two treatments were compared in terms of satisfactory efficacy. Results A total of 33 patients were included in the study, including 19 in the upadacitinib group and 14 in the abrocitinib group. At 12 weeks after treatment, EASI, VAS, IGA and BSA scores in both the upadacitinib and abrocitinib groups showed significant declines. Furthermore, there was no significant difference between the upadacitinib and abrocitinib groups in the percentage of patients achieving satisfactory outcomes. In terms of adverse reactions, 7 patients in the upadacitinib group and 5 patients in the abrocitinib group had adverse reactions, and the most common adverse reactions were acne and gastrointestinal discomfort respectively. Conclusion The results of this study indicate that JAK1 inhibitors upadacitinib and abrocitinib have good short-term efficacy and high safety in the treatment of atopic dermatitis of the head and neck. [ABSTRACT FROM AUTHOR]