- Viral discovery in the invasive Australian cane toad (Rhinella marina) using metatranscriptomic and genomic approaches. [Journal Article]
- JVJ Virol 2018 Jun 13
- Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. Release of pathogenic cane toad viruses is one possible biocontrol strat...
Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. Release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly-described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile and amphibian specific-cluster of the Picornaviridae basal to the Kobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, R. marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains eight complete RMERV proviruses, as well as 21 additional truncated insertions. The oldest full length RMERV provirus was estimated to have inserted 1.9 MYA. To screen for these viral sequences in additional toads, we analysed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, whilst RMERV transcripts were observed at all six sites. Lastly, we scanned the cane toad genome for non-retroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.Importance Cane toads are poisonous amphibians which were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threat many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality, yet few cane toad viruses have been characterised. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences which were genetically similar to viruses infecting frogs, reptiles and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies which investigate their prevalence and potential as agents for biocontrol.
- A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins α3β1, αVβ3, and αVβ5. [Journal Article]
- JVJ Virol 2018 Jun 13
- Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infec...
Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins α3β1, αVβ3, αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCE Our data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.
- LncITPRIP-1 Positively Regulates Innate Immune Response through Promoting Oligomerization and Activation of MDA5. [Journal Article]
- JVJ Virol 2018 Jun 13
- Emerging evidence indicates that long non-coding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and interfero...
Emerging evidence indicates that long non-coding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and interferon (IFN) pathway remains largely unknown. Here, we report that lncRNA ITPRIP-1 (lncITPRIP-1) is involved in viral infection and plays a crucial role in virus-triggered IFN signaling pathway through targeting MDA5. LncITPRIP-1 can be induced by viral infection, which is not entirely dependent on IFN signal. Besides, there is no coding potential found in lncITPRIP-1 transcript. LncITPRIP-1 binds to the C-terminal of MDA5 and it possesses the ability to boost oligomerization of both full length and 2CARD domains of MDA5 in a K63-linked-polyubiquitination-independent manner. Amazingly, we also find that MDA5 could suppress HCV replication independent of IFN signaling through its C-terminal deficient domain bound to viral RNA, in which lncITPRIP-1 plays as an assistant. In addition, the expression of lncITPRIP-1 is highly consistent with MDA5 expression, indicating that lncITPRIP-1 may function as a cofactor of MDA5. All the data suggest that lncITPRIP-1 enhances innate immune response to viral infection through promoting oligomerization and activation of MDA5. Our study discovers the first lncRNA ITPRIP-1 involved in MDA5 activation.SIGNIFICANCE Hepatitis C virus infection causes a global health issue and there is still no available vaccine, which makes it urgent to reveal the underlying mechanisms of HCV and host factors. Although RIG-I has been recognized as the leading cytoplasmic sensor against HCV for a long time, recent findings of MDA5 regulating IFN response to HCV have emerged. Our work validates the significant role of MDA5 in IFN signaling and HCV infection, and proposes the first lncRNA inhibiting HCV replication by promoting the activation of MDA5 and mediating the association between MDA5 and HCV RNA, which may shed light on MDA5 function study and the treatment for hepatitis C patients. Our suggested model of how lncITPRIP-1 can orchestrate signal transduction for IFN production illustrates the essential role of lncRNAs in virus elimination.
- Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis. [Journal Article]
- JVJ Virol 2018 Jun 13
- UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus HSV-1 and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been r...
UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus HSV-1 and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been reported, there is a lack of information on how these viral protein kinases are regulated in HSV-infected cells. In this study, we used a large-scale phosphoproteomic analysis of HSV-2-infected cells to identify a physiological phosphorylation site in HSV-2 UL13 (i.e., Ser-18), and investigated the significance of phosphorylation of this site in HSV-2-infected cell cultures and mice. Our results were as follows. (i) An alanine substitution at UL13 Ser-18 (S18A) significantly reduced HSV-2 replication and cell-cell spread in U2OS cells to a level similar to the UL13 null and kinase-dead mutations. (ii) The UL13 S18A mutation significantly impaired phosphorylation of a cellular substrate of this viral protein kinase in HSV-2-infected U2OS cells. (iii) Following vaginal infection of mice, the UL13 S18A mutation significantly reduced mortality, HSV-2 replication in the vagina and development of vaginal disease to levels similar to the UL13 null and the kinase-dead mutations. (iv) A phosphomimetic substitution at UL13 Ser-18 significantly restored the phenotype observed with the UL13 S18A mutation in U2OS cells and mice. Collectively, our results suggested that phosphorylation of UL13 Ser-18 regulated UL13 function in HSV-2-infected cells and this regulation was critical for the functional activity of HSV-2 UL13 in vitro and in vivo, and also for HSV-2 replication and pathogenesis.IMPORTANCE: Based on studies on cellular protein kinases, it is obvious that the regulatory mechanisms of protein kinases are as crucial as their functional consequences. Herpesviruses each encode at least one protein kinase, but the mechanism by which these kinases are regulated in infected cells remains to be elucidated with a few exceptions, although information on their functional effects has been accumulating. In this study, we have shown that phosphorylation of the HSV-2 UL13 protein kinase at Ser-18 regulated its function in infected cells, and this regulation was critical for HSV-2 replication and pathogenesis in vivo. UL13 is conserved in all members of the family Herpesviridae, and this is the first report clarifying the regulatory mechanism of a conserved herpesvirus protein kinase that is involved in viral replication and pathogenesis in vivo. Our study may provide insight into the regulatory mechanisms of the other conserved herpesvirus protein kinases.
- Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9. [Journal Article]
- JVJ Virol 2018 Jun 13
- Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiot...
Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it's largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.IMPORTANCEIn the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use.
- A single mutation at position 156 in envelope protein of Tembusu virus is responsible for virus tissue tropism and transmissibility in ducks. [Journal Article]
- JVJ Virol 2018 Jun 13
- Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV) and Bagaza virus (BAGV), is able to transmit vector-independently. T...
Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV) and Bagaza virus (BAGV), is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks.Author summaryTembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.
- Design of Novel HIV-1/2 Fusion Inhibitors with Efficient Therapeutic Efficacy in Rhesus Monkey Models. [Journal Article]
- JVJ Virol 2018 Jun 13
- T-20 (enfuvirtide) is the only approved viral fusion inhibitor, which is used for treatment of HIV-1 infection; however, it has relatively low antiviral activity and easily induces drug resistance. W...
T-20 (enfuvirtide) is the only approved viral fusion inhibitor, which is used for treatment of HIV-1 infection; however, it has relatively low antiviral activity and easily induces drug resistance. We recently reported a T-20-based lipopeptide fusion inhibitor (LP-40) showing improved anti-HIV activity (Ding et al J Virol, 2017). In this study, we designed LP-50 and LP-51 by refining the structure and function of LP-40. Two new lipopeptides showed dramatically enhanced secondary structures and binding stability and were exceptionally potent inhibitors of HIV-1, HIV-2, simian immunodeficiency virus (SIV), and simian-human chimeric virus (SHIV), with mean IC50 values at very low picomolar concentrations. They also exhibited dramatically increased potencies in inhibiting a panel of T-20 and LP-40-resistant mutant viruses. In line with their in vitro data, LP-50 and LP-51 exhibited extremely potent and long-lasting ex vivo anti-HIV activities in rhesus monkeys, with serum dilution peaks that inhibited 50% virus infection being >15,200-fold higher than T-20 and LP-40. A low-dose, short-term monotherapy of LP-51 could sharply reduce viral loads to undetectable levels in acutely and chronically SHIV-infected monkey models. In our knowledge, LP-50 and LP-51 are the most potent and broad HIV-1/2 and SIV fusion inhibitors, which can be developed for clinical use and serve as tools to explore the mechanisms of viral entry and inhibition.IMPORTANCE T-20 remains the only membrane fusion inhibitor available for treatment of viral infection, but its relatively low anti-HIV and genetic barrier for drug resistance have significantly limited its clinical application. Herein, we report two new lipopeptide-based fusion inhibitors (LP-50 and LP-51) showing extremely potent inhibitory activities against diverse HIV-1, HIV-2, SIV, and T-20-resistant variants. Promisingly, both inhibitors exhibited a potent and long-lasting ex vivo anti-HIV activity and could efficiently suppress viral loads at undetectable levels in SHIV-infected monkey models. We believe that they are the most potent and broad-spectrum fusion inhibitors known to date and thus have high potential for clinical development.
- A dual motif in the hemagglutinin of H5N1 Goose/Guangdong-like HPAIV is conserved from their early evolution and increases both membrane fusion pH and virulence. [Journal Article]
- JVJ Virol 2018 Jun 13
- Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by acquisition of a poly...
Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required co-adaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R or 124I each, in dual combination however at 97.5%. This highly conserved dual motif is still retained in contemporary H5 HPAIV including the novel H5NX reassortants carrying neuraminidases of different subtypes like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/97 carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S/124T, characteristic in low-pathogenic strains, prevented virus rescue while the single substitutions attenuated the virus both in chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less acidic endosomes, e.g. within the respiratory tract, resulting in an extended organ tropism. Taken together, HA co-adaptation to increased acid sensitivity promoted early evolution of H5 Goose/Guangdong-like HPAIV and is still required for their zoonotic potential.IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required co-adaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic in low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less acidic endosomes, e.g. within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted emergence of H5 Goose/Guangdong-like HPAIV and is required for their zoonotic potential.
- Porcine reproductive and respiratory syndrome virus infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff. [Journal Article]
- JVJ Virol 2018 Jun 13
- Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Induci...
Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest.IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2α (eIF2α). However, PRRSV nsp2 does not induce eIF2α phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by dephosphorylating eIF2α or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff.
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- Expression of a structural protein of the mycovirus FgV-ch9 negatively affects the transcript level of a novel symptom alleviation factor and causes virus-infection like symptoms in Fusarium graminearum. [Journal Article]
- JVJ Virol 2018 Jun 13
- Infections of fungi by mycoviruses are often symptomless but sometimes also fatal as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycovir...
Infections of fungi by mycoviruses are often symptomless but sometimes also fatal as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful mean to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a dsRNA chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of Fusarium Head Blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring high amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus-infection like symptoms when expressed in the wild-type but not in the vr1-overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps to understand the underlying mechanism of hypovirulence.IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful mean to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus-infection like symptoms while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e. vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.