Replication Of Wild-type Virus Research Articles

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Sp1 facilitates continued HSV-1 gene expression in the absence of key viral transactivators.

Productive replication of herpes simplex virus (HSV) relies upon a well-ordered transcriptional cascade flowing from immediate-early (IE) to early (E) to late (L) gene products. While several virus-encoded transcriptional activators are involved in this process, IE and E gene promoters also contain multiple binding sites for the ubiquitously expressed cellular transcription factor Sp1. Sp1 has been previously implicated in activating HSV-1 gene transcription downstream of these sites, but why Sp1-binding sites are maintained in the promoters of genes activated by virus-encoded activators remains unclear. We hypothesized that Sp1 enables continued HSV-1 transcription and replication when viral transactivators are limited. We used a depletion-based approach in human foreskin fibroblasts to investigate the specific contribution of Sp1 to the initiation and progression of the HSV-1 lytic gene cascade. We found that Sp1 increased viral transcript levels, protein expression, and replication following infection with VP16- or ICP0-deficient viruses but had little to no effect on rescued viruses or during wild-type (WT) HSV-1 infection. Moreover, Sp1 promoted WT virus transcription and replication following interferon treatment of fibroblasts and thus may contribute to viral immune evasion. Interestingly, we observed reduced expression of Sp1 and Sp1-family transcription factors in differentiated sensory neurons compared to undifferentiated cells, suggesting that reduced Sp1 levels may also contribute to HSV-1 latent infection. Overall, these findings indicate that Sp1 can promote HSV-1 gene expression in the absence of key viral transactivators; thus, HSV-1 may use Sp1 to maintain its gene expression and replication under adverse conditions.IMPORTANCEHerpes simplex virus (HSV) is a common human pathogen that actively replicates in the epithelia but can persist for the lifetime of the infected host via a stable, latent infection in neurons. A key feature of the HSV replication cycle is a complex transcriptional program in which virus and host-cell factors coordinate to regulate expression of the viral gene products necessary for continued viral replication. Multiple binding sites for the cellular transcription factor Sp1 are located in the promoters of HSV-1 genes, but how Sp1 binding contributes to transcription and replication of wild-type virus is not fully understood. In this study, we identified a specific role for Sp1 in maintaining HSV-1 gene transcription under adverse conditions, as when virus-encoded transcriptional activators were absent or limited. Preservation of Sp1-binding sites in HSV-1 gene promoters may thus benefit the virus as it navigates diverse cell types and host-cell conditions during infection.

Defective Interfering Particles of Influenza Virus and Their Characteristics, Impacts, and Use in Vaccines and Antiviral Strategies: A Systematic Review.

Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.

Peptidic defective interfering gene nanoparticles against Omicron, Delta SARS-CoV-2 variants and influenza A virus in vivo

Defective interfering genes (DIGs) are short viral genomes and interfere with wild-type viral replication. Here, we demonstrate that the new designed SARS-CoV-2 DIG (CD3600) can significantly inhibit the replication of SARS-CoV-2 including Alpha, Delta, Kappa and Omicron variants in human HK-2 cells and influenza DIG (PAD4) can significantly inhibit influenza virus replication in human A549 cells. One dose of influenza DIGs prophylactically protects 90% mice from lethal challenge of A(H1N1)pdm09 virus and CD3600 inhibits SARS-CoV-2 replication in hamster lungs when DIGs are administrated to lungs one day before viral challenge. To further investigate the gene delivery vector in the respiratory tract, a peptidic TAT2-P1&LAH4, which can package genes to form small spherical nanoparticles with high endosomal escape ability, is demonstrated to dramatically increase gene expression in the lung airway. TAT2-P1&LAH4, with the dual-functional TAT2-P1 (gene-delivery and antiviral), can deliver CD3600 to significantly inhibit the replication of Delta and Omicron SARS-CoV-2 in hamster lungs. This peptide-based nanoparticle system can effectively transfect genes in lungs and deliver DIGs to inhibit SARS-CoV-2 variants and influenza virus in vivo, which provides the new insight into the drug delivery system for gene therapy against respiratory viruses.

A defined anthocyanin mixture sourced from bilberry and black currant inhibits Measles virus and various herpesviruses

BackgroundAnthocyanin-containing plant extracts and carotenoids, such as astaxanthin, have been well-known for their antiviral and anti-inflammatory activity, respectively. We hypothesised that a mixture of Ribes nigrum L. (Grossulariaceae) (common name black currant (BC)) and Vaccinium myrtillus L. (Ericaceae) (common name bilberry (BL)) extracts (BC/BL) with standardised anthocyanin content as well as single plant extracts interfered with the replication of Measles virus and Herpesviruses in vitro.MethodsWe treated cell cultures with BC/BL or defined single plant extracts, purified anthocyanins and astaxanthin in different concentrations and subsequently infected the cultures with the Measles virus (wild-type or vaccine strain Edmonston), Herpesvirus 1 or 8, or murine Cytomegalovirus. Then, we analysed the number of infected cells and viral infectivity and compared the data to non-treated controls.ResultsThe BC/BL extract inhibited wild-type Measles virus replication, syncytia formation and cell-to-cell spread. This suppression was dependent on the wild-type virus-receptor-interaction since the Measles vaccine strain was unaffected by BC/BL treatment. Furthermore, the evidence was provided that the delphinidin-3-rutinoside chloride, a component of BC/BL, and purified astaxanthin, were effective anti-Measles virus compounds. Human Herpesvirus 1 and murine Cytomegalovirus replication was inhibited by BC/BL, single bilberry or black currant extracts, and the BC/BL component delphinidin-3-glucoside chloride. Additionally, we observed that BC/BL seemed to act synergistically with aciclovir. Moreover, BC/BL, the single bilberry and black currant extracts, and the BC/BL components delphinidin-3-glucoside chloride, cyanidin-3-glucoside, delphinidin-3-rutinoside chloride, and petunidin-3-galactoside inhibited human Herpesvirus 8 replication.ConclusionsOur data indicate that Measles viruses and Herpesviruses are differentially susceptible to a specific BC/BL mixture, single plant extracts, purified anthocyanins and astaxanthin. These compounds might be used in the prevention of viral diseases and in addition to direct-acting antivirals, such as aciclovir.

Flock house virus as a vehicle for aphid Virus-induced gene silencing and a model for aphid biocontrol approaches

Due to their high specificity and efficacy, RNA interference (RNAi)-based strategies have been used for fundamental functional genomics studies in a number of insects. However, its potential for translational applications in pest management is also of great interest. The lack of suitable RNAi triggering approaches, however, so far has largely precluded the implementation of RNAi-based approaches to target aphids. In this work, we first demonstrate that Flock House virus (FHV), an insect virus, can infect multiple aphid species, including the green peach aphid, Myzus persicae (M. persicae), the corn leaf aphid, Rhopalosiphum maidis (R. maidis), and the bird cherry-oat aphid, Rhopalosiphum padi (R. padi), by both microinjection and oral feeding. Using green fluorescent protein (GFP) as an indicator, we showed that the defective interfering RNA (DI-634) of FHV RNA2, which is generated autonomously during wild-type (WT) virus replication, can carry foreign sequences, and further for their functional expression. More importantly, the engineered DI-634 was incorporated into virus particles in co-infections with WT FHV. Using FHV virions containing genetically modified DI-634, the accumulation levels of the M. persicae mRNAs for Cathepsin L (CatL) and Sugar Transporter 4 (ST4), were decreased by ~ 35% and ~ 30–50%, respectively when virions were injected intrathoracically into aphids. Finally, and of more practical relevance, oral acquisition of these engineered FHV virions caused lethality of M. persicae. In summary, as a proof-of-concept, our work demonstrates that FHV can be a valuable RNAi tool for fundamental research, and suggests opportunities for using engineered insect viruses as biological agents for aphid pest control.

A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a widespread viral disease that has led to huge economic losses for the global swine industry. Non-structural protein 9 (Nsp9) of PRRSV possesses essential RNA-dependent RNA polymerase (RdRp) activity for viral RNA replication. Our previous report showed that Nsp9-specific nanobody, Nb6, was able to inhibit PRRSV replication. In this study, recombinant Nsp9 and Nsp9-Nb6 complex were prepared then characterized using bio-layer interferometry (BLI) and dynamic light scattering (DLS) analyses that demonstrated high-affinity binding of Nb6 to Nsp9 to form a homogeneous complex. Small-angle X-ray scattering (SAXS) characterization analyses revealed that spatial interactions differed between Nsp9 and Nsp9-Nb6 complex molecular envelopes. Enzyme-linked immunosorbent assays (ELISAs) revealed key involvement of Nsp9 residues Ile588, Asp590, and Leu643 and Nb6 residues Tyr62, Trp105, and Pro107 in the Nsp9-Nb6 interaction. After reverse genetics-based techniques were employed to generate recombinant Nsp9 mutant viruses, virus replication efficiencies were assessed in MARC-145 cells. The results revealed impaired viral replication of recombinant viruses bearing I588A and L643A mutations as compared with replication of wild type virus, as evidenced by reduced negative-strand genomic RNA [(−) gRNA] synthesis and attenuated viral infection. Moreover, the isoleucine at position 588 of Nsp9 was conserved across PRRSV genotypes. In conclusion, structural analysis of the Nsp9-Nb6 complex revealed novel amino acid interactions involved in viral RNA replication that will be useful for guiding development of structure-based anti-PRRSV agents.

The evolution of collective infectious units in viruses

Viruses frequently spread among cells or hosts in groups, with multiple viral genomes inside the same infectious unit. These collective infectious units can consist of multiple viral genomes inside the same virion, or multiple virions inside a larger structure such as a vesicle. Collective infectious units deliver multiple viral genomes to the same cell simultaneously, which can have important implications for viral pathogenesis, antiviral resistance, and social evolution. However, little is known about why some viruses transmit in collective infectious units, whereas others do not. We used a simple evolutionary approach to model the potential costs and benefits of transmitting in a collective infectious unit. We found that collective infectious units could be favoured if cells infected by multiple viral genomes were significantly more productive than cells infected by just one viral genome, and especially if there were also efficiency benefits to packaging multiple viral genomes inside the same infectious unit. We also found that if some viral sequences are defective, then collective infectious units could evolve to become very large, but that if these defective sequences interfered with wild-type virus replication, then collective infectious units were disfavoured.

Binding of cellular nucleolin with the viral core RNA G-quadruplex structure suppresses HCV replication.

Hepatitis C virus (HCV) infection is a major cause of human chronic liver disease and hepatocellular carcinoma. G-quadruplex (G4) is an important four-stranded secondary structure of nucleic acids. Recently, we discovered that the core gene of HCV contains a G4 RNA structure; however, the interaction between the HCV core RNA G4 and host cellular proteins, and the roles of the HCV core RNA G4 in HCV infection and pathogenesis remain elusive. Here, we identified a cellular protein, nucleolin (NCL), which bound and stabilized the HCV core RNA G4 structure. We demonstrated the direct interaction and colocalization between NCL and wild-type core RNA G4 at both in vitro and in cell physiological conditions of the alive virus; however no significant interaction was found between NCL and G4-modified core RNA. NCL is also associated with HCV particles. HCV infection induced NCL mRNA and protein expression, while NCL suppressed wild-type viral replication and expression, but not G4-modified virus. Silencing of NCL greatly enhanced viral RNA replication. Our findings provide new insights that NCL may act as a host factor for anti-viral innate immunity, and binding of cellular NCL with the viral core RNA G4 structure is involved in suppressing HCV replication.

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes

Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells. Despite a production yield of 1–2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%–70% of that produced using pAdHelper. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3–7 times higher than that using pAdHelper. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.

The Interferon-Induced Exonuclease ISG20 Exerts Antiviral Activity through Upregulation of Type I Interferon Response Proteins.

Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3'-5' exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20-/- mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle.IMPORTANCE The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20-/- mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.

Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope

BackgroundThe 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form “rafts” that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion.MethodsA large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics.ResultsVLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1–4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations.ConclusionsThis study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

Serotype-specific restriction of wild-type adenoviruses by the cellular Mre11-Rad50-Nbs1 complex

During viral replication in the nucleus, the DNA genomes of adenoviruses are accessible to cellular DNA-binding proteins. Human adenovirus type 5 (Ad5) targets the cellular Mre11-Rad50-Nbs1 complex (MRN) to evade detection by the DNA damage response (DDR). Ad5 mutants that cannot target MRN have reduced viral propagation. Previous studies showed that diverse adenovirus serotypes interact differently with MRN. While these studies revealed diverse MRN interactions among serotypes, it remains unclear how these differences influence viral replication. Here, we examined effects of the DDR on several adenovirus serotypes. We demonstrate that wild-type Ad9 and Ad12 do not overcome MRN impairment. We also examined viral proteins involved in targeting MRN and found that unlike Ad5-E4orf3, expression of Ad9-E4orf3 is not sufficient for MRN mislocalization observed during infection. We conclude that adenovirus serotypes target MRN in distinct ways, and the MRN complex can impair DNA replication of wild-type viruses across the adenovirus family.

Drug-like property-driven optimization of 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors against rilpivirine-resistant mutant virus

On the basis of our prior structure-activity relationship (SAR) results, our current lead optimization of 1,5-diarylanilines (DAANs) focused on the 4-substituent (R1) on the central phenyl ring as a modifiable position related simultaneously to improved drug resistance profiles and drug-like properties. Newly synthesized p-cyanovinyl-DAANs (8a–8g) with different R1 side chains plus prior active p-cyanoethyl-DAANs (4a–4c) were evaluated not only for anti-HIV potency against both wild-type HIV virus and rilpivirine-resistant (E138K, E138K+M184I) viral replication, but also for multiple drug-like properties, including aqueous solubility, lipophilicity, and metabolic stability in human liver microsomes and human plasma. This study revealed that both ester and amide R1 substituents led to low nanomolar anti-HIV potency against wild-type and rilpivirine-resistant viral strains (E138K-resistance fold changes<3). The N-substituted amide-R1 side chains were superior to ester-R1 likely due to improved aqueous solubility, lipophilicity, and higher metabolic stability in vitro. Thus, three amide-DAANs 8e, 4a, and 4b were identified with high potency against wild-type and rilpivirine-resistant viral strains and multiple desirable drug-like properties.

Suramin is a potent inhibitor of Chikungunya and Ebola virus cell entry.

BackgroundChikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity.MethodsWe tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus.ResultsSuramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection.ConclusionSuramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.

Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation.

Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.

Correction: Distinct Domains within the Human Cytomegalovirus UL26 Protein Are Important for Wildtype Viral Replication and Virion Stability

Correction: Distinct Domains within the Human Cytomegalovirus UL26 Protein Are Important for Wildtype Viral Replication and Virion Stability

Phosphorylation of herpes simplex virus 1 dUTPase upregulated viral dUTPase activity to compensate for low cellular dUTPase activity for efficient viral replication.

We recently reported that herpes simplex virus 1 (HSV-1) protein kinase Us3 phosphorylated viral dUTPase (vdUTPase) at serine 187 (Ser-187) to upregulate its enzymatic activity, which promoted HSV-1 replication in human neuroblastoma SK-N-SH cells but not in human carcinoma HEp-2 cells. In the present study, we showed that endogenous cellular dUTPase activity in SK-N-SH cells was significantly lower than that in HEp-2 cells and that overexpression of cellular dUTPase in SK-N-SH cells increased the replication of an HSV-1 mutant with an alanine substitution for Ser-187 (S187A) in vdUTPase to the wild-type level. In addition, we showed that knockdown of cellular dUTPase in HEp-2 cells significantly reduced replication of the mutant vdUTPase (S187A) virus but not that of wild-type HSV-1. Furthermore, the replacement of Ser-187 in vdUTPase with aspartic acid, which mimics constitutive phosphorylation, and overexpression of cellular dUTPase restored viral replication to the wild-type level in cellular dUTPase knockdown HEp-2 cells. These results indicated that sufficient dUTPase activity was required for efficient HSV-1 replication and supported the hypothesis that Us3 phosphorylation of vdUTPase Ser-187 upregulated vdUTPase activity in host cells with low cellular dUTPase activity to produce efficient viral replication.virus. Importance: It has long been assumed that dUTPase activity is important for replication of viruses encoding a dUTPase and that the viral dUTPase (vdUTPase) activity was needed if host cell dUTPase activity was not sufficient for efficient viral replication. In the present study, we showed that the S187A mutation in HSV-1 vdUTPase, which impaired its enzymatic activity, reduced viral replication in SK-N-SH cells, which have low endogenous cellular dUTPase activity, and that overexpression of cellular dUTPase restored viral replication to the wild-type level. We also showed that knockdown of cellular dUTPase in HEp-2 cells, which have higher dUTPase activity than do SK-N-SH cells, reduced replication of HSV-1 with the vdUTPase mutation but had no effect on wild-type virus replication. This is the first report, to our knowledge, directly showing that dUTPase activity is critical for efficient viral replication and that vdUTPase compensates for low host cell dUTPase activity to produce efficient viral replication.