Antiviral Approaches Research Articles

Unraveling the interplay between norovirus infection, gut microbiota, and novel antiviral approaches: a comprehensive review.

Norovirus infection is a leading cause of acute gastroenteritis worldwide and can also cause harmful chronic infections in individuals with weakened immune systems. The role of the gut microbiota in the interactions between the host and noroviruses has been extensively studied. While most past studies were conducted in vitro or focused on murine noroviruses, recent research has expanded to human noroviruses using in vivo or ex vivo human intestinal enteroids culture studies. The gut microbiota has been observed to have both promoting and inhibiting effects on human noroviruses. Understanding the interaction between noroviruses and the gut microbiota or probiotics is crucial for studying the pathogenesis of norovirus infection and its potential implications, including probiotics and vaccines for infection control. Recently, several clinical trials of probiotics and norovirus vaccines have also been published. Therefore, in this review, we discuss the current understanding and recent updates on the interactions between noroviruses and gut microbiota, including the impact of norovirus on the microbiota profile, pro-viral and antiviral effects of microbiota on norovirus infection, the use of probiotics for treating norovirus infections, and human norovirus vaccine development.

An Update on Current Antiviral Strategies to Combat Human Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) remains an essential global concern due to its distinct life cycle, mutations and latency. As HCMV is a herpesvirus, it establishes a lifelong persistence in the host through a chronic state of infection. Immunocompromised individuals are at risk of significant morbidity and mortality from the virus. Until now, no effective vaccine has been developed to combat HCMV infection. Only a few antivirals targeting the different stages of the virus lifecycle and viral enzymes are licensed to manage the infection. Therefore, there is an urgent need to find alternate strategies to combat the infection and manage drug resistance. This review will provide an insight into the clinical and preclinical antiviral approaches, including HCMV antiviral drugs and nucleic acid-based therapeutics.

Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Preparing for the next viral threat with broad-spectrum antivirals.

There is a large global unmet need for the development of countermeasures to combat hundreds of viruses known to cause human disease and for the establishment of a therapeutic portfolio for future pandemic preparedness. Most approved antiviral therapeutics target proteins encoded by a single virus, providing a narrow spectrum of coverage. This, combined with the slow pace and high cost of drug development, limits the scalability of this direct-acting antiviral (DAA) approach. Here, we summarize progress and challenges in the development of broad-spectrum antivirals that target either viral elements (proteins, genome structures, and lipid envelopes) or cellular proviral factors co-opted by multiple viruses via newly discovered compounds or repurposing of approved drugs. These strategies offer new means for developing therapeutics against both existing and emerging viral threats that complement DAAs.

TMPRSS2 Inhibitor Discovery Facilitated through an In Silico and Biochemical Screening Platform.

The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a promising antiviral target because it plays a role in priming the spike protein before viral entry occurs for the most virulent variants. Further, TMPRSS2 has no established physiological role, thereby increasing its attractiveness as a target for antiviral agents. Here, we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we identify new noncovalent TMPRSS2 inhibitors that block SARS-CoV-2 infectivity in a cellular model. One such inhibitor, debrisoquine, has high ligand efficiency, and an initial structure-activity relationship study demonstrates that debrisoquine is a tractable hit compound for TMPRSS2.

Targeting the Host Mitochondria as a Novel Human Cytomegalovirus Antiviral Strategy.

Human cytomegalovirus (HCMV) exploits host mitochondrial function to promote viral replication. HCMV gene products have been described to directly interact and alter functional or structural aspects of host mitochondria. Current antivirals against HCMV, such as ganciclovir and letermovir, are designed against viral targets. Concerns with the current antivirals include toxicity and viral resistance. Targeting host mitochondrial function is a promising alternative or complimentary antiviral approach as (1) drugs targeting host mitochondrial function interact with host targets, minimizing viral resistance, and (2) host mitochondrial metabolism plays key roles in HCMV replication. This review describes how HCMV alters mitochondrial function and highlights pharmacological targets that can be exploited for novel antiviral development.

Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann–Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.

Role of RNA Domain Structure and Orientation in the Coxsackievirus B3 Virulence Phenotype.

Coxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5' untranslated region (5' UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5' UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5' UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5' UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5' UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5' UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5' UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA. IMPORTANCE Human enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5' untranslated region (5' UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5' UTR. We present RNA structure models that directly compare the 5' UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.

RNase P Ribozyme Effectively Inhibits Human CC-Chemokine Receptor 5 Expression and Human Immunodeficiency Virus 1 Infection

Developing novel antiviral agents and approaches is essential for the treatment against human and zoonotic viruses. We had previously produced RNase P-based ribozyme variants capable of efficiently cleaving mRNA in vitro. Here, engineered ribozymes were constructed from an RNase P ribozyme variant to target the mRNA encoding human CC-chemokine receptor 5 (CCR5), an HIV co-receptor. The constructed ribozyme efficiently cleaved the CCR5 mRNA in vitro. In cells expressing the engineered ribozyme, CCR5 expression diminished by more than 90% and the infection of HIV (R5 strain Ba-L) decreased by 200-fold. The ribozyme-expressing cells resistant to R5 strain Ba-L still supported the infection of HIV X4 strain IIIB due to its use of CXCR4 instead of CCR5 as the co-receptor. Thus, the ribozyme is specific against CCR5 but not CXCR4. This indicates that RNase P ribozyme is effective and specific against CCR5 to diminish HIV infection, and also displays the viability of developing engineered RNase P ribozymes against human and zoonotic viruses.

Advances in deciphering the interactions between viral proteins of influenza A virus and host cellular proteins.

Influenza A virus (IAV) poses a severe threat to the health of animals and humans. The genome of IAV consists of eight single-stranded negative-sense RNA segments, encoding ten essential proteins as well as certain accessory proteins. In the process of virus replication, amino acid substitutions continuously accumulate, and genetic reassortment between virus strains readily occurs. Due to this high genetic variability, new viruses that threaten animal and human health can emerge at any time. Therefore, the study on IAV has always been a focus of veterinary medicine and public health. The replication, pathogenesis, and transmission of IAV involve intricate interplay between the virus and host. On one hand, the entire replication cycle of IAV relies on numerous proviral host proteins that effectively allow the virus to adapt to its host and support its replication. On the other hand, some host proteins play restricting roles at different stages of the viral replication cycle. The mechanisms of interaction between viral proteins and host cellular proteins are currently receiving particular interest in IAV research. In this review, we briefly summarize the current advances in our understanding of the mechanisms by which host proteins affect virus replication, pathogenesis, or transmission by interacting with viral proteins. Such information about the interplay between IAV and host proteins could provide insights into how IAV causes disease and spreads, and might help support the development of antiviral drugs or therapeutic approaches.

Abietane Diterpenoids Isolated from Torreya nucifera Disrupt Replication of Influenza Virus by Blocking the Phosphatidylinositol-3-Kinase (PI3K)-Akt and ERK Signaling Pathway.

Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera exhibited strong anti-influenza activity, with 50% inhibitory concentration values of 13.6 and 18.3 μM against H1N1, 12.8 and 10.8 μM against H9N2, and 29.2 μM (only compound 2) against H3N2 in the post-treatment assay, respectively. During the viral replication stages, the two compounds demonstrated stronger inhibition of viral RNA and protein in the late stages (12-18 h) than in the early stages (3-6 h). Moreover, both compounds inhibited PI3K-Akt signaling, which participates in viral replication during the later stages of infection. The ERK signaling pathway is also related to viral replication and was substantially inhibited by the two compounds. In particular, the inhibition of PI3K-Akt signaling by these compounds inhibited viral replication by sabotaging influenza ribonucleoprotein nucleus-to-cytoplasm export. These data indicate that compounds 1 and 2 could potentially reduce viral RNA and viral protein levels by inhibiting the PI3K-Akt signaling pathway. Our results suggest that abietane diterpenoids isolated from T. nucifera may be potent antiviral candidates for new influenza therapies.

Threat of respiratory syncytial virus infection knocking the door: a proposed potential drug candidate through molecular dynamics simulations, a future alternative.

The discovery of antiviral approaches to prevent or cure respiratory syncytial virus (RSV) infections is critical, particularly because RSV is one of the most common causes of infant respiratory problems. There is currently no approved vaccination available to treat RSV infections. FDA has approved the drug ribavirin, but it is not sufficient to treat RSV. This work aimed to find and study in silico anti-RSV drugs that target matrix protein and nucleoprotein. In this study, we have identified five drug candidates that had better binding energies than ribavirin. Garenoxacin appeared as top lead compounds between them. AutoDock Vina was used to execute molecular docking of a library of chosen chemicals. The high-score compound was then confirmed using the Maestro 12.3 module's molecular dynamics simulation and the binding energies derived using Prime/Molecular Mechanics Generalized Born Surface Area (Prime/MM-GBSA). Comparative molecular dynamics simulations revealed that garenoxacin has better stability and high residue contacts with high binding affinity than ribavirin. This study showed garenoxacin could prevent RSV infection better than ribavirin. In pursuing a more effective RSV control drug, additional research into these chemicals in vitro and in vivo is essential.

Antiviral Approaches against Influenza Virus.

Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.

Repurposing cholesterol lowering drugs in the treatment and management of monkeypox.

Dear Editor, While the likelihood of monkeypox (MPX) becoming a pandemic, very little is known about the virus. The smallpox-like symptoms caused by the MPX virus are less severe. Given the current possibility of a pandemic, it is crucial that any negative impacts of a MPX epidemic on public health be kept to a minimum. At the time of writing this letter, Centre for Disease control and Prevention has reported 77,092 confirmed cases across the globe in more than 89 countries. According to Centre for Disease control and Prevention, there is currently no cure for MPX infections; nevertheless, most patients recover after receiving supportive care, such as rehydration. MPX virus and Smallpox virus share genetic similarity and there is evidence to suggest that smallpox vaccination provides some protection against MPX, leading to a more favorable clinical presentation. To prevent high risk individuals, two vaccines namely JYNNEOS and ACAM2000 are used against MPX1. Among several pharmacological interventions suggested, antiviral drugs Brincidofovir and Tecovirimat show promising therapeutic effects. It inhibits the production of protein required for extracellular virus production2. Clinical trials of these antivirals are currently underway and may take a longtime to decipher its effectiveness and safety. Learning from coronavirus disease-2019 (COVID-19) pandemic, several clinical trials and observational studies have shown that statins can protect against death and reduce the symptoms of respiratory infections3. In addition to this, cholesterol lowering drugs (fenofibrates and PCSK9 inhibitors) also presented beneficial effects in COVID-19 patients with hypercholesterolemia4,5. These drugs exhibit antiviral activity by altering cell membrane cholesterol and non–lipid-related pleiotropic effects such as anti-inflammatory and immunomodulatory effects6. The entry of fusogenic, enveloped viruses into host cells requires lipid envelope, especially cholesterol. HSV and HIV-1 requires cholesterol on the host membrane and on the viral envelope for its entry. In envelope glycoprotein clustering of HIV-1, cholesterol is a major molecular platform regulating this process7. Cellular cholesterol depletion by statins reduced the production of human parainfluenza virus type 1 (hPIV1) particles by interfering virus assembly8. It also affects the stability, density of virions of influenza A virus and respiratory syncytial virus9. Lovastain and gemfibrozil inhibited hP1V1 virus assembly production through disruption of lipid raft integrity. Dengue virus infection induced the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), which decreases the recycling of low-density lipoprotein receptor to promote cholesterol redistribution into ER repressing ER-resident STING and type I interferon activation10. To combat this, PCSK9 inhibitors significantly improved the secretion of type I interferons. Statins are also repurposed in treating the patients following Ebola and Influenza infections. Overall it is clear that, statins exhibits antiviral effects by blocking/altering cholesterol trafficking, redistribution, virus attachment, virions production, maturation, release, and virions stability. MPX seems to become another worldwide public health crisis, evoking parallels to the ongoing COVID-19 epidemic and its forebears. Repurposing cholesterol lowering drugs could be an effective approach in MPX. However, there is a lacune in understanding the role of cholesterol and its redistribution in MPX. Reducing cholesterol seems to be an effective antiviral approach, it could also dampen the host’s immune system11. One must also keep in mind that since viruses rely on lipid flux, employing statins to reduce cholesterol levels that is needed for viral maintenance rather than global reduction could be an effective approach. Hypercholesterolemic patients infected with MPX should continue taking cholesterol lowering drugs. Administering PCSK9 inhibitors, statins and fenofibrates could be considered as an adjuvant therapy along with standard drugs employed in in MPX treatment. Ethical approval Not applicable. Sources of funding None. Author contribution S.S., S.K.R.S. performed literature search and drafted the letter. Conflicts of interest disclosure The authors S.S. and S.K.R.S. certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Research registration unique identifying number (UIN) None. Guarantor None.

An Insight into Emerging Begomoviruses and their Satellite Complex causing Papaya Leaf Curl Disease.

Papaya leaf curl disease (PaLCD) was primarily detected in India and causes major economic damage to agriculture crops grown globally, seriously threatening food security. Begomoviruses are communicated by the vector Bemisia tabaci, and their transmission efficiency and persistence in the vector are the highest, exhibiting the widest host range due to adaptation and evolution. Symptoms induced during PaLCD include leaf curl, leaf yellowing, interveinal chlorosis, and reduced fruit quality and yield. Consequently, plants have evolved several multi-layered defense mechanisms to resist Begomovirus infection and distribution. Subsequently, Begomovirus genomes organise circular ssDNA of size ~2.5-2.7 kb of overlapping viral transcripts and carry six-seven ORFs encoding multifunctional proteins, which are precisely evolved by the viruses to maintain the genome-constraint and develop complex but integrated interactions with a variety of host components to expand and facilitate successful infection cycles, i.e., suppression of host defense strategies. Geographical distribution is continuing to increase due to the advent and evolution of new Begomoviruses, and sweep to new regions is a future scenario. This review summarizes the current information on the biological functions of papaya-infecting Begomoviruses and their encoded proteins in transmission through vectors and modulating host-mediated responses, which may improve our understanding of how to challenge these significant plant viruses by revealing new information on the development of antiviral approaches against Begomoviruses associated with PaLCD.

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis.

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

Non-Nucleoside Inhibitors Decrease Foot-and-Mouth Disease Virus Replication by Blocking the Viral 3Dpol.

Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in the Picornaviridae family. RNA-dependent RNA polymerase (RdRp) of RNA viruses is highly conserved. Compounds that bind to the RdRp active site can block viral replication. Herein, we combined double virtual screenings and cell-based antiviral approaches to screen and identify potential inhibitors targeting FMDV RdRp (3Dpol). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3Dpol active sites. Using the BHK-21 cell-based assay, we found that four compounds-NSC217697 (quinoline), NSC670283 (spiro compound), NSC292567 (nigericin), and NSC65850-demonstrated dose-dependent antiviral actions in vitro with the EC50 ranging from 0.78 to 3.49 µM. These compounds could significantly block FMDV 3Dpol activity in the cell-based 3Dpol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV's main protease, 3Cpro. The 3Dpol inhibition activities of the compounds were consistent with the decreased viral load and negative-stranded RNA production in a dose-dependent manner. Conclusively, we have identified potential FMDV 3Dpol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment.

Targeted protein degradation as an antiviral approach

Targeted protein degradation (TPD) has emerged as a new modality in drug discovery. In this approach, small molecules are used to drive degradation of the target protein of interest. Whereas most direct-acting antivirals (DAAs) inhibit or derange the activity of their viral protein targets and have occupancy-driven pharmacology, small molecules with a TPD-based mechanism have event-driven pharmacology exerted through their ability to induce target degradation. These contrasting mechanisms can result in significant differences in drug efficacy and pharmacodynamics that may be useful in the development of new classes of antivirals. While now being widely pursued in cancer biology and autoimmune disease, TPD has not yet been widely applied as an antiviral strategy. Here, we briefly review TPD pharmacology along with the current status of tools available for developing small molecules that achieve antiviral activity through a TPD mechanism. We also highlight aspects of TPD that may be especially useful in the development of antivirals and that we hope will motivate pursuit of TPD-based antivirals by the antivirals research community.

Activity, Template Preference, and Compatibility of Components of RNA Replicase of Eastern Equine Encephalitis Virus.

Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1-4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.

Forty years of investment in HIV research: progress towards ending the HIV pandemic and preparation for future pandemics.

Forty years of investment in HIV research: progress towards ending the HIV pandemic and preparation for future pandemics.