Correction: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

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

Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3.

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145-3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

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.

A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.

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.

Early Challenges in the Implementation of Automated CranialRebuild Freeware for Generation of Patient-Specific Cranial Implant Using Additive Manufacturing: A Pilot Project in Review.

Traumatic Brain Injury (TBI) is a significant global health concern, particularly in low- and middle-income countries (LMICs) where access to medical resources is limited. Decompressive craniectomy (DHC) is a common procedure to alleviate elevated intracranial pressure (ICP) following TBI, but the cost of subsequent cranioplasty can be prohibitive, especially in resource-constrained settings. We describe challenges encountered during the beta-testing phase of CranialRebuild 1.0, an automated software program tasked with creating patient-specific cranial implants (PSCIs) from CT images. Two pilot clinical teams in the Philippines and Ukraine tested the software, providing feedback on its functionality and challenges encountered. The constructive feedback from the Philippine and Ukrainian teams highlighted challenges related to CT scan parameters, DICOM file arrays, software limitations, and the need for further software improvements. CranialRebuild 1.0 shows promise in addressing the need for affordable PSCIs in LMICs. Challenges and improvement suggestions identified throughout the beta-testing phase will shape the development of CranialRebuild 2.0, with the aim of enhancing its functionality and usability. Further research is needed to validate the software's efficacy in a clinical setting and assess its cost-effectiveness.

Clinically Isolated ß-Lactam-Resistant Gram-Negative Bacilli in a Philippine Tertiary Care Hospital Harbor Multi-Class ß-Lactamase Genes.

In the Philippines, data are scarce on the co-occurrence of multiple ß-lactamases (BLs) in clinically isolated Gram-negative bacilli. To investigate this phenomenon, we characterized BLs from various ß-lactam-resistant Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa isolated from a Philippine tertiary care hospital. The selected Gram-negative bacilli (n = 29) were resistant to either third-generation cephalosporins (resistance category 1 (RC1)), cephalosporins and penicillin-ß-lactamase inhibitors (RC2), or carbapenems (RC3). Isolates resistant to other classes of antibiotics but susceptible to early-generation ß-lactams were also selected (RC4). All isolates underwent antibiotic susceptibility testing, disk-diffusion-based BL detection assays, and PCR with sequence analysis of extended-spectrum BLs (ESBLs), metallo-BLs, AmpC BLs, and oxacillinases. Among the study isolates, 26/29 harbored multi-class BLs. All RC1 isolates produced ESBLs, with blaCTX-M as the dominant (19/29) gene. RC2 isolates produced ESBLs, four of which harbored blaTEM plus blaOXA-1 or other ESBL genes. RC3 isolates carried blaNDM and blaIMP, particularly in three of the metallo-BL producers. RC4 Enterobacteriaceae carried blaCTX-M, blaTEM, and blaOXA-24-like, while A. baumannii and P. aeruginosa in this category carried either blaIMP or blaOXA-24. Genotypic profiling, in complement with phenotypic characterization, revealed multi-class BLs and cryptic metallo-BLs among ß-lactam-resistant Gram-negative bacilli.

Phage Revolution Against Multidrug-Resistant Clinical Pathogens in Southeast Asia.

Southeast Asia (SEA) can be considered a hotspot of antimicrobial resistance (AMR) worldwide. As recent surveillance efforts in the region reported the emergence of multidrug-resistant (MDR) pathogens, the pursuit of therapeutic alternatives against AMR becomes a matter of utmost importance. Phage therapy, or the use of bacterial viruses called bacteriophages to kill bacterial pathogens, is among the standout therapeutic prospects. This narrative review highlights the current understanding of phages and strategies for a phage revolution in SEA. We define phage revolution as the radical use of phage therapy in infectious disease treatment against MDR infections, considering the scientific and regulatory standpoints of the region. We present a three-phase strategy to encourage a phage revolution in the SEA clinical setting, which involves: (1) enhancing phage discovery and characterization efforts, (2) creating and implementing laboratory protocols and clinical guidelines for the evaluation of phage activity, and (3) adapting regulatory standards for therapeutic phage formulations. We hope that this review will open avenues for scientific and policy-based discussions on phage therapy in SEA and eventually lead the way to its fullest potential in countering the threat of MDR pathogens in the region and worldwide.

Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies.

BACKGROUND: Dengue virus (DENV) infection remains a global public health concern. Enzyme-linked immunosorbent assays (ELISAs), which detect antibodies targeting the envelope (E) protein of DENV, serve as the front-line serological test for presumptive dengue diagnosis. Very few studies have determined the serostatus by detecting antibodies targeting the nonstructural protein 1 (NS1), which can function as diagnostic biomarkers to distinguish natural immunity from vaccine-induced immunity. METHODS: We used community-acquired human serum specimens, with the serostatus confirmed by focus reduction microneutralization test (FRµNT), to evaluate the diagnostic performances of two NS1-based ELISA methods, namely, immunoglobulin G antibody-capture ELISA (NS1 GAC-ELISA) and indirect NS1 IgG ELISA, and compared the results with an E-based virus-like particle (VLP) GAC-ELISA. RESULTS: NS1-based methods had comparable accuracies as VLP GAC-ELISA. Although the sensitivity in detecting anti-NS1 IgM was poor, indirect NS1 IgG ELISA showed similar limits of detection (~1-2 ng/mL) as NS1 GAC-ELISA in detecting anti-NS1 IgG. Combining the results from two or more tests as a composite reference standard can determine the DENV serostatus with a specificity reaching 100%. CONCLUSION: NS1-based ELISAs have comparable accuracies as VLP GAC-ELISA in determining dengue serostatus, which could effectively assist clinicians during assessments of vaccine eligibility.

High-resolution functional profiling of hepatitis C virus genome.

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.

Cys-diabody quantum dot conjugates (immunoQdots) for cancer marker detection.

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.

On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates.

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.

SNAP-tagged Chikungunya Virus Replicons Improve Visualisation of Non-Structural Protein 3 by Fluorescence Microscopy.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP-tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus).

Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research.

Chikungunya virus (CHIKV) is becoming an increasing global health issue which has spread across the globe and as far north as southern Europe. There is currently no vaccine or anti-viral treatment available. Although there has been a recent increase in CHIKV research, many of these in vitro studies have used a wide range of cell lines which are not physiologically relevant to CHIKV infection in vivo. In this study, we aimed to evaluate a panel of cell lines to identify a subset that would be both representative of the infectious cycle of CHIKV in vivo, and amenable to in vitro applications such as transfection, luciferase assays, immunofluorescence, western blotting and virus infection. Based on these parameters we selected four mammalian and two mosquito cell lines, and further characterised these as potential tools in CHIKV research.

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

UNLABELLED: 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.

Fidelity of target site duplication and sequence preference during integration of xenotropic murine leukemia virus-related virus.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity.