Production Of Infectious Virus Research Articles

Zinc Influx Restricts Enterovirus D68 Replication

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that causes severe respiratory diseases and neurologic manifestations. Since the 2014 outbreak, EV-D68 has been reported to cause severe complications worldwide. However, there are currently no approved antiviral agents or vaccines for EV-D68. In this study, we found that zinc ions exerted substantial antiviral activity against EV-D68 infection in vitro. Zinc salt treatment potently suppressed EV-D68 RNA replication, protein synthesis, and infectious virion production and inhibited cytopathic effects without producing significant cytotoxicity at virucidal concentrations (EC50=0.033mM). Zinc chloride (ZnCl2) treatment moderately inhibited EV-D68 attachment. Time-dose analysis of EV-D68 structural protein VP1 synthesis showed stronger suppression of VP1 in the culture medium than that in the cell lysates. Furthermore, a zinc ionophore, pyrrolidine dithiocarbamate, which can transport zinc ions into cells, also enhanced the anti-EV-D68 activity of ZnCl2 treatment. Taken together, our results demonstrated that the enhancement of zinc influx could serve as a powerful strategy for the therapeutic treatment of EV-D68 infections.

Epigenetic targeting of the ACE2 and NRP1 viral receptors limits SARS-CoV-2 infectivity

BackgroundSARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors for entry into cells, and the serine protease TMPRSS2 for S protein priming. Inhibition of protease activity or the engagement with ACE2 and NRP1 receptors has been shown to be an effective strategy for blocking infectivity and viral spreading. Valproic acid (VPA; 2-propylpentanoic acid) is an epigenetic drug approved for clinical use. It produces potent antiviral and anti-inflammatory effects through its function as a histone deacetylase (HDAC) inhibitor. Here, we propose VPA as a potential candidate to tackle COVID-19, in which rapid viral spread and replication, and hyperinflammation are crucial elements.ResultsWe used diverse cell lines (HK-2, Huh-7, HUVEC, Caco-2, and BEAS-2B) to analyze the effect of VPA and other HDAC inhibitors on the expression of the ACE-2 and NRP-1 receptors and their ability to inhibit infectivity, viral production, and the inflammatory response. Treatment with VPA significantly reduced expression of the ACE2 and NRP1 host proteins in all cell lines through a mechanism mediated by its HDAC inhibitory activity. The effect is maintained after SARS-CoV-2 infection. Consequently, the treatment of cells with VPA before infection impairs production of SARS-CoV-2 infectious viruses, but not that of other ACE2- and NRP1-independent viruses (VSV and HCoV-229E). Moreover, the addition of VPA 1 h post-infection with SARS-CoV-2 reduces the production of infectious viruses in a dose-dependent manner without significantly modifying the genomic and subgenomic messenger RNAs (gRNA and sg mRNAs) or protein levels of N protein. The production of inflammatory cytokines (TNF-α and IL-6) induced by TNF-α and SARS-CoV-2 infection is diminished in the presence of VPA.ConclusionsOur data showed that VPA blocks three essential processes determining the severity of COVID-19. It downregulates the expression of ACE2 and NRP1, reducing the infectivity of SARS-CoV-2; it decreases viral yields, probably because it affects virus budding or virions stability; and it dampens the triggered inflammatory response. Thus, administering VPA could be considered a safe treatment for COVID-19 patients until vaccines have been rolled out across the world.

Viral polymerase binding and broad-spectrum antiviral activity of molnupiravir against human seasonal coronaviruses

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, β-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.

Arginyl-tRNA-protein transferase 1 contributes to governing optimal stability of the human immunodeficiency virus type 1 core

BackgroundThe genome of human immunodeficiency virus type 1 (HIV-1) is encapsulated in a core consisting of viral capsid proteins (CA). After viral entry, the HIV-1 core dissociates and releases the viral genome into the target cell, this process is called uncoating. Uncoating of HIV-1 core is one of the critical events in viral replication and several studies show that host proteins positively or negatively regulate this process by interacting directly with the HIV-1 CA.ResultsHere, we show that arginyl-tRNA-protein transferase 1 (ATE1) plays an important role in the uncoating process by governing the optimal core stability. Yeast two-hybrid screening of a human cDNA library identified ATE1 as an HIV-1-CA-interacting protein and direct interaction of ATE1 with Pr55gag and p160gag − pol via HIV-1 CA was observed by cell-based pull-down assay. ATE1 knockdown in HIV-1 producer cells resulted in the production of less infectious viruses, which have normal amounts of the early products of the reverse transcription reaction but reduced amounts of the late products of the reverse transcription. Interestingly, ATE1 overexpression in HIV-1 producer cells also resulted in the production of poor infectious viruses. Cell-based fate-of-capsid assay, a commonly used method for evaluating uncoating by measuring core stability, showed that the amounts of pelletable cores in cells infected with the virus produced from ATE1-knockdown cells increased compared with those detected in the cells infected with the control virus. In contrast, the amounts of pelletable cores in cells infected with the virus produced from ATE1-overexpressing cells decreased compared with those detected in the cells infected with the control virus.ConclusionsThese results indicate that ATE1 expression levels in HIV-1 producer cells contribute to the adequate formation of a stable HIV-1 core. These findings provide insights into a novel mechanism of HIV-1 uncoating and revealed ATE1 as a new host factor regulating HIV-1 replication.Graphic abstract

Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir.

The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

G1-like PB2 gene improves virus replication and competitive advantage of H9N2 virus.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

Increased capsid oligomerization is deleterious to dengue virus particle production.

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis.

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein.

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

A Novel Small Molecule Inhibits Hepatitis C Virus Propagation in Cell Culture.

ABSTRACTHepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear.IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.

Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication.

Coronaviruses rely on host membranes for entry, establishment of replication centers, and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we test small molecule inhibitors that target the PI3 kinase VPS34 or fatty acid metabolism for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. Our studies determine that compounds targeting VPS34 are potent SARS-CoV-2 inhibitors. Mechanistic studies with compounds targeting multiple steps up- and downstream of fatty acid synthase (FASN) identify the importance of triacylglycerol production and protein palmitoylation as requirements for efficient viral RNA synthesis and infectious virus production. Further, FASN knockout results in significantly impaired SARS-CoV-2 replication that can be rescued with fatty acid supplementation. Together, these studies clarify roles for VPS34 and fatty acid metabolism in SARS-CoV-2 replication and identify promising avenues for the development of countermeasures against SARS-CoV-2.

Investigating Virus-Host Interactions in Cultured Primary Honey Bee Cells.

Simple SummaryHoney bees are eusocial insects that live in colonies comprised of ~30,000 individuals. They are the primary pollinators of plants that produce fruits, nuts, and vegetables. High annual losses of honey bee colonies have made it challenging for beekeepers to provide enough colonies to meet the demand for pollination services. Multiple stressors, including viruses, contribute to colony deaths. To better understand viral pathogenesis and bee antiviral defense mechanisms, we obtained primary cells from honey bee larvae and pupae, maintained them in culture, and infected them with a panel of viruses. We determined that larval hemocytes, which are primary immune cells, support replication of a honey bee virus (sacbrood virus) and a model virus (Flock House virus). Similarly, we determined that mixed cell populations derived from honey bee pupae support replication of sacbrood virus, Flock House virus, and another honey bee virus (deformed wing virus). Evaluation of the host cellular level response to infections with each of these viruses revealed unique expression profiles of three immune genes. In summary, this study demonstrates the utility of honey bee primary cell cultures to investigate the impacts of virus infection on honey bees at the cellular level, which in turn affects individual bee and colony health.Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.

HIV-1 viral blips are associated with repeated and increasingly high levels of cell-associated HIV-1 RNA transcriptional activity.

Objective:Some HIV+ patients, virally suppressed on ART, show occasional ‘blips’ of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without ‘blips’.Design/methods:RNA and DNA extracted from cells in 6 ml of peripheral blood, from suppressed patients with one to two ‘blip’ episodes over the past 2 years of ART (n = 55), or no ‘blips’ (n = 52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved ‘R’ region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus.Results:HIV-1 RNA transcripts ranged from 1.3 to 5415 copies/106 white blood cells. ‘Blip’ patients had significantly higher levels vs. without blips (median 192 vs. 49; P = 0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1.Conclusion:Viral ‘blips’ are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.

Narciclasine Display Preferential Pre-Clinical Activity Against KSHV-Associated Primary Effusion Lymphoma By Targeting MYC Via NF-κB

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of PEL. KSHV-encoded viral FLICE-inhibitory protein (vFLIP)-K13 constitutively activates the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. In a K13-isogenic cell line based screening of small molecule libraries, we have identified the natural products narciclasine and its structural analog, 7-deoxynarciclasine (found in daffodils), as having preferential cytotoxicity towards K13-expressing isogenic cell line. Further, the structurally closely related lycorine (another alkaloid found in daffodils) also exhibited similar activity. All the three compounds (Narciclasine, 7-deoxynarciclasine, and lycorine) display preferential cytotoxicity against PEL in a panel of cell lines comprising 6 hematological malignancies. Narciclasine is approximately 10 and 100-fold much effective than its structural analogs, 7-deoxynarciclasine, and lycorine, respectively. Importantly, IC50 of narciclasine in PEL cell lines (⁓10 nM) are well below the physiological concentration that was achieved in murine pharmacokinetic studies (250 nM). Narciclasine arrested cell cycle progression of PEL cells at the G0/G1 phase, with a concomitant decrease in the percentage of cells in S-phase. Further, there is a significant increase in the number of cells in sub-G0 peak, representative of apoptotic cells, which was confirmed by activation of caspase-3/7, AnnexinV staining, and PARP cleavage. Narciclasine blocked the NF-κB promoter activity in PEL cells as observed by a drastic decrease in firefly luciferase activity of PEL cells stably transduced with a lentiviral NF-κB-Luc reporter. IL-6 and IL-10 are known NF-κB targets and growth factors that are constitutively secreted by PEL, striking decrease in their secretory levels were observed upon treatment with narciclasine. Narciclasine preferentially blocked K13-induced NF-κB over the constitutive NF-κB activity in cells, which were engineered to express K13 conditionally (tet-on inducible lentiviral system). IKKγ/NEMO is essential for K13-induced NF-κB. The preference of narciclasine for K13-induced NF-κB is completely abolished upon NEMO knockout by CRISPR-Cas9 system. While probing the effect of narciclasine on the levels of putative NF-κB target genes by qRT-PCR in PEL, we found that there is a significant decrease in mRNA level of MYC, with a concomitant decrease in its protein level. Subsequently, all the direct target genes of MYC (MYB, TYRO3, TERT, PMM2, SLC19A1, and HK2) were significantly down regulated upon treatment with narciclasine. Furthermore, ectopic expression of MYC from a retroviral promoter significantly diminished the anti-proliferative effect of narciclasine and its analogs in PEL. It has been shown that inhibition of MYC is toxic to PEL and compounds that target MYC display selective activity against PEL, which explains the preferential activity of narciclasine and its analogs in PEL. MYC is required for maintenance of KSHV latency, and its down regulation results in the induction of KSHV Replication and Transcription Activator (RTA/ORF50) in PEL. However, narciclasine neither induced the expression of RTA nor the production of infectious KSHV virions in PEL. Most importantly, narciclasine doubled the survival of NSG mice bearing orthotopic - Cell Line Xenograft (CLX) and Patient Derived Xenograft (PDX) models of PEL. Finally, the IC50 of narciclasine in PBMCs from normal healthy donors is approximately 4-fold higher than the PEL cells directly isolated from the mice bearing the PDX model of the disease and PEL cell lines, indicating that there is a significant therapeutic window for narciclasine, which can be exploited for the treatment of KSHV-associated malignancies. DisclosuresChaudhary:University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript.

RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

Zika virus employs the host antiviral RNase L protein to support replication factory assembly

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

Povidone-Iodine Attenuates Viral Replication in Ocular Cells: Implications for Ocular Transmission of RNA Viruses.

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1β) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

Light and electron microscopy imaging unveils new aspects of the antiviral capacity of silver nanoparticles in bunyavirus-infected cells

Drug repurposing is an important source of new antivirals because many compounds used to treat a variety of pathologies also hamper viral infections. Habitually, silver nanoparticles (AgNPs) have been used to treat bacterial and fungal infections and their antiviral properties have been also reported. In this work, we have studied the antiviral capacity of AgNPs in cells infected with Bunyamwera virus (BUNV), the prototype of the Bunyavirales order. This group of viruses contains important pathogens for humans, animals and plants. Incubation of BUNV-infected Vero cells with non-toxic concentrations of AgNPs, reduced the production of extracellular infectious viruses in up to three orders of magnitude. With a combination of imaging techniques, we have visualized the intracellular distribution of AgNPs in mock- and BUNV-infected cells and studied their effects on intracellular organelles. In mock-infected cells and at short times post-incubation, AgNPs were detected inside nuclei and mitochondria by transmission electron microscopy (TEM). At long times post-treatment, they accumulated inside lysosome-like organelles. Cell compartments did not exhibit any appreciable ultrastructural alterations after incubation with AgNPs. In BUNV-infected cells, AgNPs attached to extracellular virions, that showed a disrupted morphology. Inside cells, they were detected inside the nucleus, in mitochondria and around characteristic Golgi-associated, single-membrane spherules. These membranous structures are the replication organelles (ROs) of bunyaviruses and contain active viral replication complexes (VRCs). Compared to normal spherules that are round, compact and have an electron-dense core, spherules in AgNPs-treated cells were deformed and their core was electron-lucent. Interestingly, in BUNV-infected cells treated with the typical antiviral ribavirin (RBV), spherules with VRCs exhibit also an anomalous morphology and an electron-lucent core. Both AgNPs and RBV might interfere with BUNV-induced dismantling of cell nucleoli and with the intercellular propagation of large groups of virions, a mechanism of BUNV transmission observed for the first time in cultured cells. Our results point to silver nanoparticles as good candidates for antiviral therapy, either alone or in combination with other antiviral drugs, such as RBV-related compounds.

The Second-Generation XPO1 Inhibitor Eltanexor Inhibits Human Cytomegalovirus (HCMV) Replication and Promotes Type I Interferon Response.

Human cytomegalovirus (HCMV) is a ubiquitous opportunistic pathogen and can be life-threatening for immunocompromised individuals. There is currently no available vaccine for the prevention of HCMV- associated diseases and most of the available antiviral drugs that target viral DNA synthesis become ineffective in treating HCMV mutants that arise after long-term use in immunocompromised patients. Here, we examined the effects of Eltanexor, a second-generation selective inhibitor of nuclear export (SINE), on HCMV replication. Eltanexor effectively inhibits HCMV replication in human foreskin fibroblasts in a dose-dependent manner. Eltanexor does not significantly inhibit viral entry and nuclear import of viral genomic DNA, but rather suppress the transcript and protein levels of viral immediate-early (IE), early (E) and late (L) genes, and abolishes the production of infectious virions. We further found Eltanexor treatment promotes proteasome-mediated degradation of XPO1, which contributes to the nuclear retention of interferon regulatory factor 3 (IRF-3), resulting in increased expression of type I interferon as well as interferon stimulating genes ISG15 and ISG54. This study reveals a novel antiviral mechanism of Eltanexor which suggests it has potential to inhibit a broad spectrum of viral pathogens.

Characterizing infection of B lymphocytes with vaccine and wild-type strains of measles virus

Abstract Wild-type (w.t.) measles virus (MeV) infection can result in immunosuppression that does not occur following vaccination. A rhesus macaque model indicated that B cells are a target of infection. The role of B cell infection in MeV-induced immune suppression is not fully understood. We characterize the differences in viral replication in human B cells infected with vaccine (Edmonston Zagreb [EZ]) or w.t. virus. B cells from healthy donor blood were infected (MOI 1.0) with a GFP-expressing EZ virus or a w.t. virus to evaluate viral replication after 24 and 48 hours. The number of GFP+ cells (EZ infected) or hemagglutinin (H)+ cells (w.t. infected) was measured via flow cytometry. Transcription of the viral nucleoprotein (N) gene was measured by mRNA-specific cDNA generation and N gene specific qPCR. Production of infectious viral particles was measured via endpoint dilution. In B cells infected with EZ, 30% of cells expressed GFP at 24 hours and 45% of cells expressed GFP at 48 hours. There was a 4.4-fold increase in N gene transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. In B cells infected with w.t. virus, 10% of cells expressed viral H at 24 hours with no increase in H expression at 48 hours. The w.t. virus had a 2.4-fold increase in viral transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. MeV-infected B cells show viral protein production and N gene transcription at 24 and 48 hours. However, w.t. virus replication may be less efficient as shown by lower percentages of H+ cells at 24 and 48 hours and lower levels of N gene transcription at 48 hours. There is no evidence of infectious virus production by either strain of MeV despite evidence of replication.