SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks.

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Mammalian ANP32A and ANP32B Proteins Drive Differential Polymerase Adaptations in Avian Influenza Virus.

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.

Network analytics for drug repurposing in COVID-19.

To better understand the potential of drug repurposing in COVID-19, we analyzed control strategies over essential host factors for SARS-CoV-2 infection. We constructed comprehensive directed protein-protein interaction (PPI) networks integrating the top-ranked host factors, the drug target proteins and directed PPI data. We analyzed the networks to identify drug targets and combinations thereof that offer efficient control over the host factors. We validated our findings against clinical studies data and bioinformatics studies. Our method offers a new insight into the molecular details of the disease and into potentially new therapy targets for it. Our approach for drug repurposing is significant beyond COVID-19 and may be applied also to other diseases.

Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A.

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

RACK1 Associates with RNA-Binding Proteins Vigilin and SERBP1 to Facilitate Dengue Virus Replication.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no effective treatment is available. DENV relies heavily on the host cellular machinery for productive infection. Here, we show that the scaffold protein RACK1, which is part of the DENV replication complex, mediates infection by binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to an RNA interference screen-identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV genome. Genetic ablation of Vigilin or SERBP1 rendered cells poorly susceptible to DENV, as well as related flaviviruses, by hampering the translation and replication steps. Finally, we established that a Vigilin or SERBP1 mutant lacking RACK1 binding but still interacting with the viral RNA is unable to mediate DENV infection. We propose that RACK1 recruits Vigilin and SERBP1, linking the DENV genome to the translation machinery for efficient infection. IMPORTANCE We recently identified the scaffolding RACK1 protein as an important host-dependency factor for dengue virus (DENV), a positive-stranded RNA virus responsible for the most prevalent mosquito-borne viral disease worldwide. Here, we have performed the first RACK1 interactome in human cells and identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV RNA to regulate viral replication. Importantly, Vigilin and SERBP1 interact with RACK1 and the DENV viral RNA (vRNA) to mediate viral replication. Overall, our results suggest that RACK1 acts as a binding platform at the surface of the 40S ribosomal subunit to recruit Vigilin and SERBP1, which may therefore function as linkers between the viral RNA and the translation machinery to facilitate infection.

The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

CRISPR-Cas system to discover host-virus interactions in Flaviviridae.

The Flaviviridae virus family members cause severe human diseases and are responsible for considerable mortality and morbidity worldwide. Therefore, researchers have conducted genetic screens to enhance insight into viral dependency and develop potential anti-viral strategies to treat and prevent these infections. The host factors identified by the clustered regularly interspaced short palindromic repeats (CRISPR) system can be potential targets for drug development. Meanwhile, CRISPR technology can be efficiently used to treat viral diseases as it targets both DNA and RNA. This paper discusses the host factors related to the life cycle of viruses of this family that were recently discovered using the CRISPR system. It also explores the role of immune factors and recent advances in gene editing in treating flavivirus-related diseases. The ever-increasing advancements of this technology may promise new therapeutic approaches with unique capabilities, surpassing the traditional methods of drug production and treatment.

SNHG15 aids SARS-CoV-2 entry via RABL2A.

Angiotensin-converting enzyme 2 (ACE2) and several proteins have been identified as entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether long noncoding RNAs are involved in SARS-CoV-2 entry remains unknown. In this study, we investigated the role of small nucleolar RNA host gene 15 (SNHG15) in SARS-CoV-2 entry using a SARS-CoV-2 spike pseudotyped lentivirus with a luciferase reporter. Overexpression of SNHG15 promoted but SNHG15 knockdown limited SARS-CoV-2 entry in a dose- and time-dependent manner. SNHG15 interacted with Rab-like protein 2A (RABL2A). Overexpression and knockdown of RABL2A produced similar effects on SARS-CoV-2 entry as those of SNHG15. Furthermore, RABL2A knockdown abolished the SNHG15-mediated increase in SARS-CoV-2 entry. In conclusion, SNHG15 is a critical regulatory factor that aids SARS-CoV-2 entry through RABL2A.

Ectoparasites associated with the Bushveld gerbil (Gerbilliscus leucogaster) and the role of the host and habitat in shaping ectoparasite diversity and infestations.

Rodents are known hosts for various ectoparasite taxa such as fleas, lice, ticks and mites. South Africa is recognized for its animal diversity, yet little is published about the parasite diversity associated with wild rodent species. By focusing on a wildlife-human/domestic animal interface, the study aims to record ectoparasite diversity and levels of infestations of the Bushveld gerbil, Gerbilliscus leucogaster, and to establish the relationship between ectoparasite infestation parameters and host- and habitat factors. Rodents (n = 127) were trapped in 2 habitat types (natural and agricultural) during 2014­2020. More than 6500 individuals of 32 epifaunistic species represented by 21 genera and belonging to 5 taxonomic groups (fleas, sucking lice, ticks, mesostigmatan mites and trombiculid mites) were collected. Mesostigmatan mites and lice were the most abundant and fleas and mesostigmatan mites the most prevalent groups. Flea and mesostigmatan mite numbers and mesostigmatan mite species richness was significantly higher on reproductively active male than female rodents. Only ticks were significantly associated with habitat type, with significantly higher tick numbers and more tick species on rodents in the natural compared to the agricultural habitat. We conclude that the level of infestation by ectoparasites closely associated with the host (fleas and mites) was affected by host-associated factors, while infestation by ectoparasite that spend most of their life in the external environment (ticks) was affected by habitat type.

A CRISPR-Cas Cure for HIV/AIDS.

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.

Relevance of HBx for Hepatitis B Virus-Associated Pathogenesis.

The hepatitis B virus (HBV) counts as a major global health problem, as it presents a significant causative factor for liver-related morbidity and mortality. The development of hepatocellular carcinomas (HCC) as a characteristic of a persistent, chronic infection could be caused, among others, by the pleiotropic function of the viral regulatory protein HBx. The latter is known to modulate an onset of cellular and viral signaling processes with emerging influence in liver pathogenesis. However, the flexible and multifunctional nature of HBx impedes the fundamental understanding of related mechanisms and the development of associated diseases, and has even led to partial controversial results in the past. Based on the cellular distribution of HBx-nuclear-, cytoplasmic- or mitochondria-associated-this review encompasses the current knowledge and previous investigations of HBx in context of cellular signaling pathways and HBV-associated pathogenesis. In addition, particular focus is set on the clinical relevance and potential novel therapeutic applications in the context of HBx.

Regulation of influenza A virus infection by Lnc-PINK1-2:5.

Influenza virus causes approximately 291,000 to 646,000 human deaths worldwide annually. It is also a disease of zoonotic importance, affecting animals such as pigs, horses, and birds. Even though vaccination is being used to prevent influenza virus infection, there are limited options available to treat the disease. Long noncoding RNAs (lncRNAs) are RNA molecules with more than 200 nucleotides that do not translate into proteins. They play important roles in the physiological and pathological processes. In this study, we identified a novel transcript, Lnc-PINK1-2:5 that was upregulated by influenza virus. This lncRNA was predominantly located in the nucleus and was not affected by type I interferons. Overexpression of Lnc-PINK1-2:5 reduced the influenza viral mRNA and protein levels in cells as well as titres in culture media. Knockdown of Lnc-PINK1-2:5 using CRISPR interference enhanced the virus replication. Antiviral activity of Lnc-PINK1-2:5 was independent of influenza virus strains. RNA sequencing analysis revealed that Lnc-PINK1-2:5 upregulated thioredoxin interacting protein (TXNIP) during influenza virus infection. Overexpression of TXNIP reduced influenza virus infection, suggesting that TXNIP is an antiviral gene. Knockdown of TXNIP abolished the Lnc-PINK1-2:5-mediated increase in influenza virus infection. In conclusion, the newly identified Lnc-PINK1-2:5 isoform is an anti-influenza lncRNA acting through the upregulation of TXNIP gene expression.

Understanding the key functions of Myosins in viral infection.

Myosins, a class of actin-based motor proteins existing in almost any organism, are originally considered only involved in driving muscle contraction, reshaping actin cytoskeleton, and anchoring or transporting cargoes, including protein complexes, organelles, vesicles. However, accumulating evidence reveals that myosins also play vital roles in viral infection, depending on viral species and infection stages. This review systemically summarizes the described various myosins, the performed functions, and the involved mechanisms or molecular pathways during viral infection. Meanwhile, the existing issues are also discussed. Additionally, the important technologies or agents, including siRNA, gene editing, and myosin inhibitors, would facilitate dissecting the actions and mechanisms for described and undescribed myosins, which could be adopted to prevent or control viral infection are also characterized.

Host factors and vaccine efficacy: Implications for COVID-19 vaccines.

The efficacy of the vaccines varies between individuals and populations. The immunogenicity of the vaccine is influenced by various factors, including host factors. Previous studies have shown that host factors affect the effectiveness of vaccines, which may be true about COVID-19 vaccines. In this review, we evaluate the possible association of host factors with vaccine efficacy with a special focus on COVID-19 vaccines.

Compartmentalized replication organelle of flavivirus at the ER and the factors involved.

Flaviviruses are positive-sense single-stranded RNA viruses that pose a considerable threat to human health. Flaviviruses replicate in compartmentalized replication organelles derived from the host endoplasmic reticulum (ER). The characteristic architecture of flavivirus replication organelles includes invaginated vesicle packets and convoluted membrane structures. Multiple factors, including both viral proteins and host factors, contribute to the biogenesis of the flavivirus replication organelle. Several viral nonstructural (NS) proteins with membrane activity induce ER rearrangement to build replication compartments, and other NS proteins constitute the replication complexes (RC) in the compartments. Host protein and lipid factors facilitate the formation of replication organelles. The lipid membrane, proteins and viral RNA together form the functional compartmentalized replication organelle, in which the flaviviruses efficiently synthesize viral RNA. Here, we reviewed recent advances in understanding the structure and biogenesis of flavivirus replication organelles, and we further discuss the function of virus NS proteins and related host factors as well as their roles in building the replication organelle.

Regulation of SARS CoV-2 host factors in the kidney and heart in rats with 5/6 nephrectomy-effects of salt, ARB, DPP4 inhibitor and SGLT2 blocker.

BACKGROUND: Host factors such as angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine-subtype-2 (TMPRSS2) are important factors for SARS-CoV-2 infection. Clinical and pre-clinical studies demonstrated that RAAS-blocking agents can be safely used during a SARS-CoV-2 infection but it is unknown if DPP-4 inhibitors or SGLT2-blockers may promote COVID-19 by increasing the host viral entry enzymes ACE2 and TMPRSS2. METHODS: We investigated telmisartan, linagliptin and empagliflozin induced effects on renal and cardiac expression of ACE2, TMPRSS2 and key enzymes involved in RAAS (REN, AGTR2, AGT) under high-salt conditions in a non-diabetic experimental 5/6 nephrectomy (5/6 Nx) model. In the present study, the gene expression of Ace2, Tmprss2, Ren, Agtr2 and Agt was assessed with qRT-PCR and the protein expression of ACE2 and TMPRSS2 with immunohistochemistry in the following experimental groups: Sham + normal diet (ND) + placebo (PBO); 5/6Nx + ND + PBO; 5/6Nx + high salt-diet (HSD) + PBO; 5/6Nx + HSD + telmisartan; 5/6Nx + HSD + linagliptin; 5/6Nx + HSD + empagliflozin. RESULTS: In the kidney, the expression of Ace2 was not altered on mRNA level under disease and treatment conditions. The renal TMPRSS2 levels (mRNA and protein) were not affected, whereas the cardiac level was significantly increased in 5/6Nx rats. Intriguingly, the elevated TMPRSS2 protein expression in the heart was significantly normalized after treatment with telmisartan, linagliptin and empagliflozin. CONCLUSIONS: Our study indicated that there is no upregulation regarding host factors potentially promoting SARS-CoV-2 virus entry into host cells when the SGLT2-blocker empagliflozin, telmisartan and the DPP4-inhibitor blocker linagliptin are used. The results obtained in a preclinical, experimental non-diabetic kidney failure model need confirmation in ongoing interventional clinical trials.

Host-Directed Antiviral Therapy.

Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.