Nicholas C. Wu

Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

ABSTRACT Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.

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.

A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic β-Hairpin Structure

&NA; Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo‐electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3‐fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer‐specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans. HighlightsApex binding antibody PGT145 engages all three gp120 protomers simultaneouslyEpitope recognition is chemical‐feature specificPGT145‐class antibodies exhibit structural features that reflect bovine antibodiesPGT145‐class antibody maturation is dependent on structural stabilization of HCDR3 &NA; Broadly neutralizing antibodies of the PGT145‐family target the HIV‐1 Env trimer apex via a long &bgr;‐hairpin HCDR3, but the molecular basis of recognition is unknown. Using cryoEM, Lee et al. (2017) reveal how PGT145 binds its quaternary epitope and the importance of HCDR2 evolution despite its lack of contacts with Env.

A structural explanation for the low effectiveness of the seasonal influenza H3N2 vaccine.

The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

Accurate viral population assembly from ultra-deep sequencing data

Motivation: Next-generation sequencing technologies sequence viruses with ultra-deep coverage, thus promising to revolutionize our understanding of the underlying diversity of viral populations. While the sequencing coverage is high enough that even rare viral variants are sequenced, the presence of sequencing errors makes it difficult to distinguish between rare variants and sequencing errors. Results: In this article, we present a method to overcome the limitations of sequencing technologies and assemble a diverse viral population that allows for the detection of previously undiscovered rare variants. The proposed method consists of a high-fidelity sequencing protocol and an accurate viral population assembly method, referred to as Viral Genome Assembler (VGA). The proposed protocol is able to eliminate sequencing errors by using individual barcodes attached to the sequencing fragments. Highly accurate data in combination with deep coverage allow VGA to assemble rare variants. VGA uses an expectation–maximization algorithm to estimate abundances of the assembled viral variants in the population. Results on both synthetic and real datasets show that our method is able to accurately assemble an HIV viral population and detect rare variants previously undetectable due to sequencing errors. VGA outperforms state-of-the-art methods for genome-wide viral assembly. Furthermore, our method is the first viral assembly method that scales to millions of sequencing reads. Availability: Our tool VGA is freely available at http://genetics.cs.ucla.edu/vga/ Contact: serghei@cs.ucla.edu; eeskin@cs.ucla.edu

Adaptation in protein fitness landscapes is facilitated by indirect paths

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. DOI: http://dx.doi.org/10.7554/eLife.16965.001

Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCE While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

A Perspective on the Structural and Functional Constraints for Immune Evasion: Insights from Influenza Virus

Influenza virus evolves rapidly to constantly escape from natural immunity. Most humoral immune responses to influenza virus target the hemagglutinin (HA) glycoprotein, which is the major antigen on the surface of the virus. The HA is composed of a globular head domain for receptor binding and a stem domain for membrane fusion. The major antigenic sites of HA are located in the globular head subdomain, which is highly tolerant of amino acid substitutions and continual addition of glycosylation sites. Nonetheless, the evolution of the receptor-binding site and the stem region on HA is severely constrained by their functional roles in engaging the host receptor and in mediating membrane fusion, respectively. Here, we review how broadly neutralizing antibodies (bnAbs) exploit these evolutionary constraints to protect against diverse influenza strains. We also discuss the emerging role of other epitopes that are conserved only in subsets of viruses. This rapidly increasing knowledge of the evolutionary biology, immunology, structural biology, and virology of influenza virus is invaluable for development and design of more universal influenza vaccines and novel therapeutics.

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.

High-Throughput Identification of Loss-of-Function Mutations for Anti-Interferon Activity in the Influenza A Virus NS Segment

ABSTRACT Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.