Yushen Du

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.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA–protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

MYC-induced reprogramming of glutamine catabolism supports optimal virus replication.

Viruses rewire host cell glucose and glutamine metabolism to meet the bioenergetic and biosynthetic demands of viral propagation. However, the mechanism by which viruses reprogram glutamine metabolism and the metabolic fate of glutamine during adenovirus infection have remained elusive. Here, we show MYC activation is necessary for adenovirus-induced upregulation of host cell glutamine utilization and increased expression of glutamine transporters and glutamine catabolism enzymes. Adenovirus-induced MYC activation promotes increased glutamine uptake, increased use of glutamine in reductive carboxylation and increased use of glutamine in generating hexosamine pathway intermediates and specific amino acids. We identify glutaminase (GLS) as a critical enzyme for optimal adenovirus replication and demonstrate that GLS inhibition decreases replication of adenovirus, herpes simplex virus 1 and influenza A in cultured primary cells. Our findings show that adenovirus-induced reprogramming of glutamine metabolism through MYC activation promotes optimal progeny virion generation, and suggest that GLS inhibitors may be useful therapeutically to reduce replication of diverse viruses.

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.

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.

HIV-1 Quasispecies Delineation by Tag Linkage Deep Sequencing

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 identification of interferon-sensitive mutations enables influenza vaccine design

Avoiding interferon avoidance Interferon (IFN) expression is a mammals first response to viral infection. Many viruses have thus evolved mechanisms to evade IFN. Du et al. developed a method to systematically ablate IFN evasion genes from live, attenuated influenza virus (see the Perspective by Teijaro and Burton). A combination of mutants was assembled to construct a virus that triggered transient IFN responses in mice but that was unable to replicate effectively. The transient IFN responses led to robust antibody and memory responses that protected against subsequent challenge with different influenza viruses. This approach could be adapted to improve other RNA virus vaccines. Science, this issue p. 290; see also p. 277 High-throughput genomics can be used to retune attenuated viruses to optimize vaccine development. In conventional attenuated viral vaccines, immunogenicity is often suboptimal. Here we present a systematic approach for vaccine development that eliminates interferon (IFN)–modulating functions genome-wide while maintaining virus replication fitness. We applied a quantitative high-throughput genomics system to influenza A virus that simultaneously measured the replication fitness and IFN sensitivity of mutations across the entire genome. By incorporating eight IFN-sensitive mutations, we generated a hyper–interferon-sensitive (HIS) virus as a vaccine candidate. HIS virus is highly attenuated in IFN-competent hosts but able to induce transient IFN responses, elicits robust humoral and cellular immune responses, and provides protection against homologous and heterologous viral challenges. Our approach, which attenuates the virus and promotes immune responses concurrently, is broadly applicable for vaccine development against other pathogens.

Coupling high-throughput genetics with phylogenetic information reveals an epistatic interaction on the influenza A virus M segment

BackgroundEpistasis is one of the central themes in viral evolution due to its importance in drug resistance, immune escape, and interspecies transmission. However, there is a lack of experimental approach to systematically probe for epistatic residues.ResultsBy utilizing the information from natural occurring sequences and high-throughput genetics, this study established a novel strategy to identify epistatic residues. The rationale is that a substitution that is deleterious in one strain may be prevalent in nature due to the presence of a naturally occurring compensatory substitution. Here, high-throughput genetics was applied to influenza A virus M segment to systematically identify deleterious substitutions. Comparison with natural sequence variation showed that a deleterious substitution M1 Q214H was prevalent in circulating strains. A coevolution analysis was then performed and indicated that M1 residues 121, 207, 209, and 214 naturally coevolved as a group. Subsequently, we experimentally validated that M1 A209T was a compensatory substitution for M1 Q214H.ConclusionsThis work provided a proof-of-concept to identify epistatic residues by coupling high-throughput genetics with phylogenetic information. In particular, we were able to identify an epistatic interaction between M1 substitutions A209T and Q214H. This analytic strategy can potentially be adapted to study any protein of interest, provided that the information on natural sequence variants is available.

Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein

Significance Understanding how viruses interact with their hosts, especially the mechanisms that restrict virus replication, will provide a molecular basis for vaccine development. However, the search for restriction factors is oftentimes difficult if the virus has already evolved to counteract the restriction. Here, we describe a systematic approach to identify such restriction and counterrestriction mechanisms. We constructed a library of mutant hepatitis C viruses, where each mutant has a 15-nt stretch randomly inserted on the genome. We aimed to identify mutations that lose the anti-IFN function, but maintain replication capacity. We have identified p7 as an immune evasion protein and further characterize the antiviral function of IFI6-16 against hepatitus C virus (HCV) replication. Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to down-regulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.

High-resolution genetic profile of viral genomes: why it matters

The approval of novel antiviral treatments for hepatitis C virus (HCV) infection provides a great example of research driven medicine in action. However, the emergence of resistant viral strains to existing treatments reminds us of the ongoing challenge that we still face in HCV therapy. What can be done to minimize the health risk posed by viral variants that develop resistance and cause failure of therapy? Here we propose that a high-resolution genetic profiling approach that can assess the function at a single nucleotide/amino acid resolution, may provide a solution. We further discuss the potential application of this methodology in resolving viral resistance through the following three aspects: (1) high-resolution mapping of inflexible regions on the viral genome to identify better drug targets; (2) exhaustive drug resistance profiles to facilitate next-generation drug design; (3) coupled with closely monitoring within-host virus quasi-species, drug resistance profiles can aid in optimized drug combination and personalized medicine in HCV treatments.