High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay

Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors(HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference(HT-RNAi) screens has led to the identification of several key factors involved in the viral lifecycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested(such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase Ⅲα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HTRNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.

Viral-host molecular interactions and metabolic modulation:Strategies to inhibit flaviviruses pathogenesis

Flaviviruses,which include globally impactful pathogens,such as West Nile virus,yellow fever virus,Zika virus,Japanese encephalitis virus,and dengue virus,contribute significantly to human infections.Despite the ongoing emergence and resurgence of flavivirus-mediated pathogenesis,the absence of specific therapeutic options remains a challenge in the prevention and treatment of flaviviral infections.Through the intricate processes of fusion,transcription,replication,and maturation,the complex interplay of viral and host metabolic interactions affects pathophysiology.Crucial interactions involve metabolic molecules,such as amino acids,glucose,fatty acids,and nucleotides,each playing a pivotal role in the replication and maturation of flaviviruses.These viral-host metabolic molecular interactions hijack and modulate the molecular mechanisms of host metabolism.A comprehensive understanding of these intricate metabolic pathways offers valuable insights,potentially unveiling novel targets for therapeutic interventions against flaviviral pathogenesis.This review emphasizes promising avenues for the development of therapeutic agents that target specific metabolic molecules,such as amino acids,glucose,fatty acids,and nucleotides,which interact with flavivirus replication and are closely linked to the modulation of host metabolism.The clinical limitations of current drugs have prompted the development of new inhibitory strategies for flaviviruses based on an understanding of the molecular interactions between the virus and the host.

病毒感染与宿主抗感染免疫之间“博弈”——凋亡、坏死和焦亡分子机制

细胞死亡是宿主抗病毒感染免疫的重要组成部分,病毒感染可引起不同形式宿主细胞死亡,包括溶解性和非溶解性两种细胞死亡类型。这两种细胞死亡类型不仅能够消除病毒感染细胞,而且还能够利用炎症因子释放进一步促进宿主先天和适应性免疫进程。反之,病毒也发展出不同规避机制来抑制宿主细胞死亡进而促进自身感染。本文就病毒感染与宿主抗病毒感染免疫之间的“博弈”——凋亡、坏死和焦亡调控机制研究进展进行综述,旨在为深入理解病毒的分子致病机制以及开发新的抗病毒策略提供参考资料。

Natural killer cells in hepatitis C:Current progress

Patients infected with the hepatitis C virus(HCV) are characterized by a high incidence of chronic infection, which results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The functional impairment of HCV-specific T cells is associated with the evolution of an acute infection to chronic hepatitis. While T cells are the important effector cells in adaptive immunity, natural killer(NK) cells are the critical effector cells in innate immunity to virus infections. The findings of recent studies on NK cells in hepatitis C suggest that NK cell responses are indeed important in each phase of HCV infection. In the early phase, NK cells are involved in protective immunity to HCV. The immune evasion strategies used by HCV may target NK cells and might contribute to the progression to chronic hepatitis C. NK cells may control HCV replication and modulate hepatic fibrosis in the chronic phase. Further investigations are, however, needed, because a considerable number of studies observed functional impairment of NK cells in chronic HCV infection. Interestingly, the enhanced NK cell responses during interferon-α-based therapy of chronic hepatitis C indicate successful treatment. In spite of the advances in research on NK cells in hepatitis C, establishment of more physiological HCV infection model systems is needed to settle unsolved controversies over the role and functional status of NK cells in HCV infection.

Neutralizing antibodies in hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Immediate–Early(IE) gene regulation of cytomegalovirus:IE1-and pp71-mediated viral strategies against cellular defenses

Three crucial hurdles hinder studies on human cytomegalovirus(HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus–host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host(latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate–early(IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate–early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.

A Minireview of Marine Algal Virus-Coccolithoviruses

Coccolithophorid is unicellular marine microalgae with a global distribution in temperate and sub-temperate oceanic regions and has the ability to produce ＇the coccoliths＇. It is considered to be the second most productive calcifying organism on earth and becoming an important factor in the global carbonate cycle. Emiliania huxleyi is one of the only two bloom-forming coccolithophores and becomes a species crucial to the study of global biogeochemical cycles and climate modeling. Coccolithoviruse is a recently discovered group of viruses infecting the marine coceolithophorid E. huxleyi. They are a major cause of coceolithophore bloom termination, and DMSP concentration is increasing in the process of viral lysis. Phylogenetic evidences support that some genes are functional both in E. huxleyi and its virus （EhV）. Horizontal gene transfer （HGT） of multiple functionally coupled enzymes occurs in E. huxleyi and its DNA virus EhV has been confirmed, which contributes to the diversification and adaptation of plankton in the oceans and also critically regulates virus-host infection by allowing viruses to control host metabolic pathways for their repli- cation. Therefore, it is of particular interest to understand this host-virus interaction. On this issue, we have made a minireview of coeeolithoviruses focusing on the basic characteristics, phylogenesis, horizontal gene transfer and the interaction between the host and its viruses, as well as its important role in global biogeochemical cycling.

Architecture and biogenesis of plus-strand RNA virus replication factories

Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virusinduced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.

松毛虫质型多角体病毒的宿主域与交叉感染

自 1956年从赤松毛虫Dendrolimusspectabilis上首次发现赤松毛虫质型多角体病毒 1型 (D .spectabiliscytovirus 1,DsCPV 1)以来 ,先后从马尾松毛虫D .punctatus、油松毛虫D .tabulaeformis、赤松毛虫、德昌松毛虫D .p .tehchangensis、文山松毛虫D .p .wenshangensis和落叶松毛虫D .superans上发现了质型多角体病毒 (cytoplasmicpolyhedrosisvirus ,CPV)。病毒基因组dsRNA电泳图谱分析表明 ,这些松毛虫CPV的不同分离株均属于质型多角体病毒 1型 (cytovirus 1)。这些松毛虫CPV病毒可以感染鳞翅目 10科 3 5种昆虫 ,其中对多种昆虫具有很高的感染力和良好的杀虫效果 ,可以从中筛选替代宿主生产松毛虫CPV杀虫剂 ,用于害虫生物防治。松毛虫CPV接种某些昆虫后病毒的基因组dsRNA电泳图谱发生了改变 。

Host preference of the major strains of Plum pox virus--Opinions based on regional and world-wide sequence data

Plum pox virus（PPV）causes sharka-the most serious viral disease of stone fruit trees.PPV is wide spread in Europe and Mediterranean Basin,its incidence has been further approved in Asia and both Americas.Nine PPV strains have been recognized until now（PPV-D,PPV-M,PPV-Rec,PPV-EA,PPV-C,PPV-T,PPV-W,PPV-CR,and PPV-An）,forming molecularly distinct entities,however,only partially differentiable by their biological or epidemiological properties.The most strict virus-host linkages under natural conditions have been detected for strains naturally infecting cherries（PPV-C and PPV-CR）.However,although less stringent but still clear host preference is observed also for three epidemiologically most important strains（PPV-D/plum/apricot,PPV-M/peach,and PPV-Rec/plum）.So far no genetic marker has been mapped in the PPV genome,which responsibility for the host specificity/preference could be explicitly demonstrated.In this review,we focus on the host preference of three major PPV strains as evidenced by analysis of an extensive dataset of PPV isolates of Slovak and world-wide origin.Together,we discuss several performed relevant experiments and further possible research procedures aimed to better understand the genetic determinants and mechanisms of the host preference of this potyvirus.

<i>Imperata yellow mottle virus</i>: An Emerging Threat to Maize, Sorghum and Pearl Millet in Burkina Faso

Imperata yellow mottle caused by Imperata yellow mottle virus (IYMV) of the genus Sobemovirus was first characterized on Imperata cylindrical and Zea mays in Burkina Faso. The disease has been reported in several localities of the country but its ecology and epidemiology are poorly known. In particular, only I. cylindrical and maize have been reported within IYMV host range. The aim of this study was to investigate the experimental host range of the virus. Mechanical inoculation of a mixture of four IYMV isolates to 18 plant species, including four crops (maize, rice, sorghum and pearl millet) and 14 wild grasses showed clear mottle symptoms in maize, sorghum and pearl millet and two wild grass species (Setaria verticillata and Rottboellia exaltata). Symptom development was confirmed by Enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction (RT-PCR). Infection of crop species by IYMV depended on cultivars tested. Therefore, proportions of positive cultivars were 16/36 in maize, 4/10 in sorghum and 4/9 in pearl millet, respectively. Studies on virus-host interactions using individual virus isolates showed two pathogenic patterns. Three out of the four isolates tested infected all plant species and cultivars. In contrast, the fourth IYMV isolate could infect only one maize cultivar. These results expand the previously known host range of IYMV from two to five species, indicating a narrow host range. Among the new characterized host species, sorghum and pearl millet are important cereal crops. Therefore, Imperata yellow motte disease is a potential threat for the cereal crop production and its ecology and epidemiology should be thoroughly investigated.

Reprogramming the host: Modification of cell functions upon viral infection

Viruses and their hosts have co-evolved for million years. In order to successfully replicate their genome, viruses need to usurp the biosynthetic machinery of the host cell. Depending on the complexity and the nature of the genome, replication might involve or not a relatively large subset of viral products, in addition to a number of host cell factors, and take place in several subcellular compartments, including the nucleus,the cytoplasm, as well as virus-induced, rearranged membranes. Therefore viruses need to ensure the correct subcellular localization of their effectors and to be capable of disguising from the cellular defensive mechanisms. In addition, viruses are capable of exploiting host cell activities, by modulating their post-translational modification apparatus, resulting in profound modifications in the function of cellular and viral products. Not surprisingly infection of host cells by these parasites can lead to alterations of cellular differentiation and growing properties, with important pathogenic consequences. In the present hot topic highlight entitled "Reprogramming the host: modification of cell functions upon viral infection", a number of leading virologists and cell biologist thoroughly describe recent advances in our understanding of how viruses modulate cellular functions to achieve successful replication and propagation at the expenses of human cells.

From Entry to Evasion:A Comprehensive Analysis of Host-Virus Interactions for Monkeypox

Monkeypox(Mpox)has posed a novel challenge and emerged as a threat to global public health since the onset of its outbreak in 2022.Mpox is spreading throughout the world in both endemic and non-endemic countries,indicating that its behavior is evolving.The prevalence of Mpox and the risk of a global pandemic necessitate a better understanding of Mpox virus replications and interactions with the host.Here,we attempted to provide a detailed comprehensive review of Mpox virus behavior at the molecular level from the entry level to the establishment of a successful infection,including attachment and entry,DNA replication,protein expression and viron assembly and egress.This review also describes its strategies to evade host immune responses and inhibit apoptosis,and uncovers underlying molecular mechanisms such as subverted signaling pathways and cellular factors behind host-viral interactions.

Nonmuscle myosin IIA promotes the internalization of influenza A virus and regulates viral polymerase activity through interacting with nucleoprotein in human pulmonary cells

Influenza A virus(IAV),responsible for seasonal epidemics and recurring pandemics,represents a global threat to public health.Given the risk of a potential IAV pandemic,it is increasingly important to better understand virushost interactions and develop new anti-viral strategies.Here,we reported nonmuscle myosin IIA(MYH9)-mediated regulation of IAV infection.MYH9 depletion caused a profound inhibition of IAV infection by reducing viral attachment and internalization in human lung epithelial cells.Surprisingly,overexpression of MYH9 also led to a significant reduction in viral productive infection.Interestingly,overexpression of MYH9 retained viral attachment,internalization,or uncoating,but suppressed the viral ribonucleoprotein(vRNP)activity in a minigenome system.Further analyses found that excess MYH9 might interrupt the formation of vRNP by interacting with the viral nucleoprotein(NP)and result in the reduction of the completed vRNP in the nucleus,thereby inhibiting subsequent viral RNA transcription and replication.Together,we discovered that MYH9 can interact with IAV NP protein and engage in the regulation of vRNP complexes,thereby involving viral replication.These findings enlighten new mechanistic insights into the complicated interface of host-IAV interactions,ultimately making it an attractive target for the generation of antiviral drugs.

Transcriptome profiling highlights regulated biological processes and type III interferon antiviral responses upon Crimean-Congo hemorrhagic fever virus infection

Crimean-Congo hemorrhagic fever virus(CCHFV)is a biosafety level-4(BSL-4)pathogen that causes Crimean-Congo hemorrhagic fever(CCHF)characterized by hemorrhagic manifestation,multiple organ failure and high mortality rate,posing great threat to public health.Despite the recently increasing research efforts on CCHFV,host cell responses associated with CCHFV infection remain to be further characterized.Here,to better understand the cellular response to CCHFV infection,we performed a transcriptomic analysis in human kidney HEK293 cells by high-throughput RNA sequencing(RNA-seq)technology.In total,496 differentially expressed genes(DEGs),including 361 up-regulated and 135 down-regulated genes,were identified in CCHFV-infected cells.These regulated genes were mainly involved in host processes including defense response to virus,response to stress,regulation of viral process,immune response,metabolism,stimulus,apoptosis and protein catabolic process.Therein,a significant up-regulation of type III interferon(IFN)signaling pathway as well as endoplasmic reticulum(ER)stress response was especially remarkable.Subsequently,representative DEGs from these processes were well validated by RT-qPCR,confirming the RNA-seq results and the typical regulation of IFN responses and ER stress by CCHFV.Furthermore,we demonstrate that not only type I but also type III IFNs(even at low dosages)have substantial anti-CCHFV activities.Collectively,the data may provide new and comprehensive insights into the virus-host interactions and particularly highlights the potential role of type III IFNs in restricting CCHFV,which may help inform further mechanistic delineation of the viral infection and development of anti-CCHFV strategies.

一种流感病毒多宿主传播的量化研究方法

目的探究甲型流感病毒宿主分布规律,量化评估其多宿主传播趋势。方法从流感病毒公共数据库中下载流感病毒全基因组数据,对编码序列进行特征提取,采用自然聚类结果对流感病毒进行宿主分布与进化态势分析,建立宿主谱熵值计算方法量化评估流感病毒多宿主传播扩散趋势,并使用机器学习方法对分析结果进行验证。结果H3N2型流感病毒多宿主分布界限清晰,具有最低宿主谱熵值;H9N2型流感病毒多宿主分布混乱,不同宿主的毒株序列相似程度高,具有最高宿主谱熵值。不同类型甲型流感病毒的宿主谱熵值大小与多宿主传播扩散趋势紧密相关。结论受传播环境与选择压力的影响,不同流感病毒的进化方向与多宿主传播趋势呈现出较为明显的差异,较为适应人类宿主的H3N2流感病毒的多宿主传播趋势更有序,而频繁发生跨宿主传播的H7N9、H9N2等禽流感病毒的多宿主传播趋势更混乱,进化方向也更多样。宿主谱熵值是衡量流感病毒多宿主传播趋势与进化方向有序性的高效计算方法,利用该方法有助于了解流感病毒的进化与多宿主分布,评估传播风险,可为流感病毒的监测和预警提供新的见解。

Diversity,evolutionary contribution and ecological roles of aquatic viruses

Aquatic viruses include infected viruses in aquatic animals, plants and microorganisms, and free-floating viruses(virioplankton)in water environments. In the last three decades, a huge number of aquatic viruses, especially diverse free-floating viruses,including cyanophages, phycoviruses, archaea viruses, giant viruses, and even virophages, have been identified by virological experiments and metagenomic analyses. Based on a comprehensive introduction of aquatic virus classification and their morphological and genetic diversity, here, we summarize and outline main virus species, their evolutionary contribution to aquatic communities through horizontal gene transfer, and their ecological roles for cyanobacterial bloom termination and global biogeochemical cycling in freshwater and marine ecosystems. Thereby, some novel insights of aquatic viruses and virus-host interactions, especially their evolutionary contribution and ecological rolesin diverse aquatic communities and ecosystems, are highlighted in this review.

Host cellular signaling induced by influenza virus

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell's innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.

MicroRNA-135a Modulates Hepatitis C Virus Genome Replication through Downregulation of Host Antiviral Factors

Cellular microRNAs(miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map of HCV–miRNA interactions through genome-wide miRNA functional screens and transcriptomics analyses. Many previously unappreciated cellular miRNAs were identified to be involved in HCV infection, including miR-135a, a human cancerrelated miRNA. In the present study, we investigated the role of miR-135a in regulating HCV life cycle and showed that it preferentially enhances viral genome replication. Bioinformatics-based integrative analyses and subsequent functional assays revealed three antiviral host factors, including receptor interacting serine/threonine kinase 2(RIPK2), myeloid differentiation primary response 88(MYD88), and C-X-C motif chemokine ligand 12(CXCL12), as bona fide targets of miR-135a. These genes have been shown to inhibit HCV infection at the RNA replication stage. Our data demonstrated that repression of key host restriction factors mediated the proviral effect of miR-135a on HCV propagation. In addition,miR-135a hepatic abundance is upregulated by HCV infection in both cultured hepatocytes and human liver, likely mediating a more favorable environment for viral replication and possibly contributing to HCV-induced liver malignancy.These results provide novel insights into HCV–host interactions and unveil molecular pathways linking miRNA biology to HCV pathogenesis.

Proteomics Profiling of Host Cell Response via Protein Expression and Phosphorylation upon Dengue Virus Infection

Dengue virus(DENV)infection is a worldwide public health threat.To date,the knowledge about the pathogenesis and progression of DENV infection is still limited.Combining global profiling based on proteomic analysis together with functional verification analysis is a powerful strategy to investigate the interplay between the virus and host cells.In the present study,quantitative proteomics has been applied to evaluate host responses(as indicated by altered proteins and modifications)in human cells(using K562 cell line)upon DENV-2 infection,as DENV-2 spreads most widely among all DENV serotypes.Comparative analysis was performed to define differentially expressed proteins in the infected cells compared to the mock-control,and it revealed critical pathogen-induced changes covering a broad spectrum of host cellular compartments and processes.We also discovered more dramatic changes(>20%,160 regulated phosphoproteins)in protein phosphorylation compared to protein expression(14%,321 regulated proteins).Most of these proteins/phosphoproteins were involved in transcription regulation,RNA splicing and processing,immune system,cellular response to stimulus,and macromolecule biosynthesis.Western blot analysis was also performed to confirm the proteomic data.Potential roles of these altered proteins were discussed.The present study provides valuable large-scale protein-related information for elucidating the functional emphasis of host cell proteins and their post-translational modifications in virus infection,and also provides insight and protein evidence for understanding the general pathogenesis and pathology of DENV.