A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

Intracellular dynamics of polydnavirus innexin homologues.

Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT-PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co-occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope-tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf-Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co-transfection of constructs indicates extensive co-localization of Vinnexins with each other and Sf-Inx2, and live-cell imaging of mCherry-labelled Inx2 supports that Vinnexins may affect Sf-Inx2 distribution in a Vinnexin-specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein-specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.

A Laterally Transferred Viral Gene Modifies Aphid Wing Plasticity.

Organisms often respond to changing environments by altering development of particular traits. These plastic traits exhibit genetic variation; i.e., genotypes respond differently to the same environmental cues. Theoretical studies have demonstrated the importance of this variation, which is targeted by natural selection, in adapting plastic responses to maximize fitness [1, 2]. However, little is known about the underlying genetic mechanisms. We identify two laterally transferred genes that contribute to variation in a classic example of phenotypic plasticity: the pea aphid's ability to produce winged offspring in response to crowding. We discovered that aphid genotypes vary extensively for this trait and that aphid genes of viral origin are upregulated in response to crowding solely in highly inducible genotypes. We knocked down expression of these genes to demonstrate their functional role in wing plasticity. Through phylogenetic analysis, we found that these genes likely originated from a virus that infects rosy apple aphids and causes their hosts to produce winged offspring [3]. The function of these genes has therefore been retained following transfer to pea aphids. Our results uncover a novel role for co-opted viral genes, demonstrating that they are used to modulate ecologically relevant, plastic phenotypes. Our findings also address a critical question about the evolution of environmentally sensitive traits: whether the genes that control the expression of plastic traits also underlie variation in plasticity. The genes we identify originated from outside aphids themselves, and thus, our work shows that genes formerly unrelated to plasticity can fine-tune the strength of plastic responses to the environment.

Phage single-gene lysis: Finding the weak spot in the bacterial cell wall.

In general, the last step in the vegetative cycle of bacterial viruses, or bacteriophages, is lysis of the host. dsDNA phages require multiple lysis proteins, including at least one enzyme that degrades the cell wall (peptidoglycan (PG)). In contrast, the lytic ssDNA and ssRNA phages have a single lysis protein that achieves cell lysis without enzymatically degrading the PG. Here, we review four "single-gene lysis" or Sgl proteins. Three of the Sgls block bacterial cell wall synthesis by binding to and inhibiting several enzymes in the PG precursor pathway. The target of the fourth Sgl, L from bacteriophage MS2, is still unknown, but we review evidence indicating that it is likely a protein involved in maintaining cell wall integrity. Although only a few phage genomes are available to date, the ssRNA Leviviridae are a rich source of novel Sgls, which may facilitate further unraveling of bacterial cell wall biosynthesis and discovery of new antibacterial agents.

Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice.

The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro, but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.

The role of CTX and RS1 satellite phages genomic arrangement in Vibrio cholera toxin production in two recent cholera outbreaks (2012 and 2013) in IR Iran.

The objective of the present study was to investigate the genomic arrangement of CTX/RS1 prophages in 30 Vibrio cholerae strains obtained from 2 consecutive years of cholera outbreak and to compare the role of different CTX/RS1 arrangements in cholera toxin expression among the El Tor strains. Profile A with TLC-RS1-CTX-RTX arrangement was observed in 46.7% of the isolates with RS1 phage locating adjacent to TLC element. About 50% of the isolates showed Profile B with TLC-CTX-RS1-RTX arrangement and one single isolate (3.3%) revealed TLC-CTX-RS1-RS1-RTX arrangement (Profile C). No RS1 element was detected to be adjacent to TLC element in B and C profiles. No truncated CTX phage genome was detected among the isolates of 2 years. Different CTX-RS1 arrangement profiles (A, B, and C) with different RS1 copy numbers and locations uniformly showed low level of cholera toxin production in El Tor strains with no significant difference, revealing that different RS1 copy numbers and locations have no effect on cholera toxin production level (p-value >0.05). However, increased cholera toxin expression was observed for control V. cholerae classical biotype strain. In conclusion, variations in RS1 prophage did not affect CT expression level in related El Tor V. cholerae strains. CTX genotyping establishes a more valuable database for epidemiologic, pathogenesis, and source tracking purposes.

Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus.

In order to gain insight into the role of the transcription regulatory sequences (TRSs) in the regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus (PRRSV), the enhanced green fluorescent protein (EGFP) gene, under the control of the different structural gene TRSs, was inserted between the N gene and 3'-UTR of the PRRSV genome and EGFP expression was analyzed for each TRS. TRSs of all the studied structural genes of PRRSV positively modulated EGFP expression at different levels. Among the TRSs analyzed, those of GP2, GP5, M, and N genes highly enhanced EGFP expression without altering replication of PRRSV. These data indicated that structural gene TRSs could be an extremely useful tool for foreign gene expression using PRRSV as a vector.

Pathogenicity of reassortant H9 influenza viruses with different NA genes in mice and chickens.

To better understand the influence of different NA genes on pathogenicity of H9 viruses, three reassortant H9 viruses (rH9N1, H9N2 and rH9N3) were generated and characterized. All three viruses replicated efficiently in eggs and MDCK cells, whereas the rH9N1 and rH9N3 replicated more efficiently than H9N2 in A549 cells. The rH9N3 replicated more efficiently than rH9N1 and H9N2 viruses in mice, however, rH9N3 replicated and shed less efficiently than the H9N2 virus in chickens. Further studies indicate that N3 had higher NA activity and released virus from erythrocytes faster, which may improve the adaptation of H9 influenza virus to mammals.

Effect of ORF119 gene deletion on the replication and virulence of orf virus.

Orf is a severe infectious disease of sheep and goats caused by orf virus (ORFV). To investigate the role of ORF119 gene of ORFV, we constructed ORFV with deleted ORF119 gene and LacZ as reporter gene (ORFV-Δ119-LacZ) via homologous recombination. The results showed that wild-type ORF-SHZ1 and ORFV-Δ119-LacZ deletion viruses replicated in Vero cells to similar titers. Relative transcriptional levels of virulence genes OVIFNR, GIF, VEGF and VIL-10 of ORFV-Δ119-LacZ deletion virus were slightly but not significantly lower after 24 hr compared with the wtORF-SHZ1 virus. In vivo experiments showed that 2-month-old lambs inoculated with ORFV-Δ119-LacZ deletion virus exhibited a similar total clinical score compared with those inoculated with wtORF-SHZ1 virus. Based on these results, we conclude that deletion of the ORF119 gene has no significant effect on ORFV replication and virulence.

Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4+ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL.

Large Tailed Spindle Viruses of Archaea: a New Way of Doing Viral Business.

Viruses of Archaea continue to surprise us. Archaeal viruses have revealed new morphologies, protein folds, and gene content. This is especially true for large spindle viruses, which infect only Archaea. We present a comparison of particle morphologies, major coat protein structures, and gene content among the five characterized large spindle viruses to elucidate defining characteristics. Structural similarities and a core set of genes support the grouping of the large spindle viruses into a new superfamily.

Identification of Spodoptera exigua nucleopolyhedrovirus genes involved in pathogenicity and virulence.

Genome sequence analysis of seven different Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolates that differed in insecticidal phenotype permitted the identification of genes likely to be involved in pathogenicity of occlusion bodies (OBs) and speed of kill (virulence) of this virus: se4 (hoar), se5 (unknown function), se28 (unknown function), se76 (cg30), se87 (p26) and se129 (p26). To study the role of these genes experimentally on the insecticidal phenotype, a bacmid-based recombination system was constructed to delete selected genes from a SeMNPV isolate, VT-SeAL1, designated as SeBacAL1. All of the knockout viruses were viable and the repair viruses behaved like the wild-type control, vSeBacAL1. Deletion of se4, se5, se76 and se129 resulted in decreased OB pathogenicity compared to vSeBacAL1 OBs. In contrast, deletion of se87 did not significantly affect OB pathogenicity, whereas deletion of se28 resulted in significantly increased OB pathogenicity. Deletion of se4, se28, se76, se87 and se129 did not affect speed of kill compared to the bacmid vSeBacAL1, whereas speed of kill was significantly extended following deletion of se5 and in the wild-type isolate (SeAL1), compared to that of the bacmid. Therefore, biological assays confirmed that several genes had effects on virus insecticidal phenotype. Se5 is an attractive candidate gene for further studies, as it affects both biological parameters of this important biocontrol virus.

The Epstein-Barr virus encoded BART miRNAs potentiate tumor growth in vivo.

The human herpes virus Epstein-Barr virus (EBV) latently infects and drives the proliferation of B lymphocytes in vitro and is associated with several forms of lymphoma and carcinoma in vivo. The virus encodes ~30 miRNAs in the BART region, the function of most of which remains elusive. Here we have used a new mouse xenograft model of EBV driven carcinomagenesis to demonstrate that the BART miRNAs potentiate tumor growth and development in vivo. No effect was seen on invasion or metastasis, and the growth promoting activity was not seen in vitro. In vivo tumor growth was not associated with the expression of specific BART miRNAs but with up regulation of all the BART miRNAs, consistent with previous observations that all the BART miRNAs are highly expressed in all of the EBV associated cancers. Based on these observations, we suggest that deregulated expression of the BART miRNAs potentiates tumor growth and represents a general mechanism behind EBV associated oncogenesis.

Bluetongue virus nonstructural protein NS3/NS3a is not essential for virus replication.

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG(Met)→GCC(Ala) mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.

[The construction of a novel recombinant virus Δ67R-RGV and preliminary analyses the function of the 67R gene].

The Rana grylio virus (RGV) is a member of the genus Ranavirus. It belongs to the family Iridoviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed Δ67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluorescence gene (EGFP) as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosum cyprini (EPC) cells were infected with the resulting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally Δ67R-RGV was generated. Total DNA of Δ67R-RGV infected cells was extracted for PCR analyses. Simulateously, mock infected and wild-type RGV (wt-RGV) infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in Δ67R-RGV infected cells. Furthermore, one-step growth curves of wt-RGV and Δ67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essential for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.

A balanced ratio of proteins from gene G and frameshift-extended gene GT is required for phage lambda tail assembly.

In bacteriophage λ, the overlapping open reading frames G and T are expressed by a programmed translational frameshift similar to that of the gag-pol genes of many retroviruses to produce the proteins gpG and gpGT. An analogous frameshift is widely conserved among other dsDNA tailed phages in their corresponding "G" and "GT" tail genes even in the absence of detectable sequence homology. The longer protein gpGT is known to be essential for tail assembly, but the requirement for the shorter gpG remained unclear because mutations in gene G affect both proteins. A plasmid system that can direct the efficient synthesis of tails was created and used to show that gpG and gpGT are both essential for correct tail assembly. Phage complementation assays under conditions where levels of plasmid-expressed gpG or gpGT could be altered independently revealed that the correct molar ratio of these two related proteins, normally determined by the efficiency of the frameshift, is also crucial for efficient assembly of functional tails. Finally, the physical connection between the G and T domains of gpGT, a consequence of the frameshift mechanism of protein expression, appears to be important for efficient tail assembly.

Abortive lytic reactivation of KSHV in CBF1/CSL deficient human B cell lines.

Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

Transposable elements as genetic regulatory substrates in early development.

The abundance and ancient origins of transposable elements (TEs) in eukaryotic genomes has spawned research into the potential symbiotic relationship between these elements and their hosts. In this review, we introduce the diversity of TEs, discuss how distinct classes are uniquely regulated in development, and describe how they appear to have been coopted for the purposes of gene regulation and the orchestration of a number of processes during early embryonic development. Although young, active TEs play an important role in somatic tissues and evolution, we focus mostly on the contributions of the older, fixed elements in mammalian genomes. We also discuss major challenges inherent in the study of TEs and contemplate future experimental approaches to further investigate how they coordinate developmental processes.

Genetic studies on the virus-like regions in the genome of hyperthermophilic archaeon, Thermococcus kodakarensis.

Four virus-like integrated elements (TKV1, TKV2, TKV3, and TKV4) have been found in the genome of hyperthermophilic archaeon, Thermococcus kodakarensis, but virus particle formation has not been observed in the culture of T. kodakarensis. As the result of growth property analyses, mutants lacking each of the four virus-like regions exhibited decrease in the cell concentration and/or less growth rates compared to growth of parental strain (KU216), when the T. kodakarensis strains were grown at 85 °C in nutrient-rich medium. These results indicated that the genes in virus-like regions stimulated the cell growth under the observed growth condition. As the result of transcriptome analyses, genes involved in amino acid, energy or nucleotide metabolisms, and transport systems were up- or down-regulated in the cells of mutant strains. Interestingly, a decrease in transcriptional levels of glutamine synthetase (TK1796) gene (Tk-glnA) was observed in the cells of four mutant strains. Growths of TKV1 disrupted strain and TKV4 disrupted strain have shown no difference compared with that of KU216 by the addition of glutamate or glutamine, and the result suggested that TKV1 and TKV4 contributed to supply of amino acids to the cell.