Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.     

Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development.     

Rhythm of a life within life: role of viral suppressors in hijacking the host cell

Virus–host interaction at cellular and intra-cellular level is a constantly evolving process which results either in the host to resist or the virus to break host immunity and to establish the disease. We have put extensive efforts to understand the genomic organization and gene functions of important viral proteins involved in resisting or avoiding host antiviral responses mediated by RNA interference or Ubiquitin–Proteasome Pathway. Nearly two decades of dedicated research on three agriculturally important viruses revealed genetic and epigenetic regulation of host to induce the defense/immunity responses. The microRNA and auxin regulated development of disease symptoms and the role of temperature in optimizing the interactions of viral protein with host small RNAs are intriguing observations. Owing to the complexity of the dynamic interactions between plant and virus, this research field will always be challenging and fascinating.

     

What Happens Inside Lentivirus or Influenza Virus Infected Cells: Insights into Regulation of Cellular and Viral Protein Synthesis

Efficient manipulation of the regulatory mechanisms controlling host cell gene expression provides the means for productive infection by animal viruses. Upon infecting the host cell, viruses must: (i) bypass the cellular antiviral defense mechanisms to prevent the translational blocks imposed by the interferon pathway; and (ii) effectively “hijack” the host protein synthetic machinery into mass production of virion protein components. The multicomponent regulatory nature of cellular gene expression has provided the means of selecting for a diverse range of mechanisms utilized by animal viruses to ensure that replication efficiency is maintained throughout the virus life cycle. One important research component of the careful examination of gene regulation is those studies that focus on elucidating the mechanisms by which viruses control mRNA translation during host cell infection. Much of the work in our laboratory has focused on elucidating the strategies by which human immunodeficiency virus type 1 and influenza virus regulate protein synthesis during infection. Here we describe the ways in which these two distinctly different RNA viruses ensure the selective and efficient translation of their viral mRNAs in infected cells. These strategies include circumvention of the deleterious effects associated with activation of the interferon-induced protein kinase, PKR. Herein we describe our methodologies designed to elucidate the translational regulation in cells infected by these viruses. We conclude with a brief summary of new directions, utilizing these methods, taken toward understanding the translational control mechanisms imposed by these viral systems, and how our studies of virally infected cells have allowed us to identify growth-regulating components of normal, uninfected cells.     

Coronavirus gene 7 counteracts host defenses and modulates virus virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus.     

Micro RNA and viral infections in mammals

RNA silencing plays an important role in development through the action of micro (mi) RNAs that fine tune the expression of a large portion of the genome. But, in plants and insects, it is also a very important player in innate immune responses, especially in antiviral defense. It is now well established that the RNA silencing machinery targets plant as well as insect viruses. While the genetic basis underlying this defense mechanism in these organisms starts being elucidated, much less is known about the possible antiviral role of RNA silencing in mammals. In order to identify siRNAs coming from viruses in infected human cells, small RNAs from cells infected with RNA viruses, such as hepatitis C virus, yellow fever virus or HIV-1, were cloned and sequenced, but no virus-specific siRNAs could be detected. On the contrary, viral small RNAs were found in cells infected by the DNA virus Epstein-Barr. A closer look at these revealed that they were not siRNAs, but rather resembled miRNAs. This finding indicated that, rather than being targeted by RNA silencing, human DNA viruses seem to have evolved their own miRNAs to modulate the expression of host genes. This primary observation has been extended to other members of the herpesvirus family as well as other DNA viruses such as the polyomavirus SV40. Viral miRNAs have the potential to act both in cis to regulate expression of viral genes, or in trans on host genes. There are good indications for the cis mode of action, but the identification of cellular targets of these small viral regulators is only in its infancy.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

A baculovirus-encoded MicroRNA (miRNA) suppresses its host miRNA biogenesis by regulating the exportin-5 cofactor Ran

MicroRNAs have emerged as key players in the regulation of various biological processes in eukaryotes, including host-pathogen interactions. Recent studies suggest that viruses encode miRNAs to manipulate their host gene expression to ensure their effective proliferation, whereas the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we demonstrate that an insect virus, Bombyx mori nucleopolyhedrosis virus (BmNPV), modulates the small-RNA-mediated defense of its host, B. mori, by encoding an miRNA (bmnpv-miR-1) that downregulates the expression of the host GTP-binding nuclear protein Ran, an essential component of the exportin-5-mediated nucleocytoplasmic transport machinery mainly involved in small-RNA transport from the nucleus to the cytoplasm. We demonstrate the sequence-dependent interaction of bmnpv-miR-1 with Ran mRNA using cell culture and in vivo assays, including RNA interference (RNAi) of Ran. Our results clearly show that bmnpv-miR-1 represses Ran, leading to reduction in the host small-RNA population, and consequently, the BmNPV load increases in the infected larvae. Blocking of bmnpv-miR-1 resulted in higher expression levels of Ran and a decrease in BmNPV proliferation. In contrast, blockage of host miRNA, bmo-miR-8, which targets the immediate-early gene of the virus and whose production was repressed upon bmnpv-miR-1 and Ran dsRNA administration, resulted in a significant increase in the virus load in the infected B. mori larvae. The present study provides an insight into one of the evasion strategies used by the virus to counter the host defense for its effective proliferation and has relevance to the development of insect virus control strategies.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions

Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.     

Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems

RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Correspondingly, plant viruses encode suppressors of silencing. In the positive-strand RNA virus Tobacco rattle virus (TRV), the suppressor of silencing is a 16-kDa (16K) protein encoded by RNA1. The suppressor action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by tomato bushy stunt virus. Mutant TRV that does not produce its suppressor, unlike other suppressor-defective viruses, is competent to accumulate and spread systemically in the infected plant. However, this mutant virus does not exhibit the transient invasion of the meristem that is characteristic of the wild-type virus. Based on this analysis, we propose that the 16K suppressor of silencing allows TRV to transiently invade the meristem. Our data are consistent with a mechanism of long-term meristem virus exclusion that is dependent on a transient invasion of the meristem early in the infection cycle. This novel mechanism of meristem exclusion may be associated with the phenomenon of recovery in virus-infected plants in which upper leaves have little or no virus and are immune to secondary infection by the same virus.     

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.     

Systematic identification of novel, essential host genes affecting bromovirus RNA replication

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ～100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ～900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ～81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.     

Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.