Epidermal growth factor receptor-dependent stimulation of differentiation by human papillomavirus type 16 E5.

Human papillomaviruses (HPVs) infect keratinocytes of stratified squamous epithelia, and persistent infection with high-risk HPV types, such as HPV16, may lead to the development of malignancies. HPV evades host immunity in part by linking its gene expression to the host differentiation program, and therefore relies on differentiation to complete its life cycle. Based on previous reports indicating that the HPV16 protein E5 is important in the late stages of the differentiation-dependent life cycle, we found that organotypic cultures harboring HPV16 genomes lacking E5 showed reduced markers of terminal differentiation compared to wild type HPV16-containing cultures. We found that epidermal growth factor receptor (EGFR) levels and activation were increased in an E5-depdendent manner in these tissues, and that EGFR promoted terminal differentiation and expression of the HPV16 L1 gene. These findings suggest a function for E5 in preserving the ability of HPV16 containing keratinocytes to differentiate, thus facilitating the production of new virus progeny.

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

Suppression of a Subset of Interferon-Induced Genes by Human Papillomavirus Type 16 E7 via a Cyclin Dependent Kinase 8-Dependent Mechanism.

Persistent infection by human papillomaviruses (HPVs), small, double-stranded DNA viruses that infect keratinocytes of the squamous epithelia, can lead to the development of cervical and other cancers. The viral oncoprotein E7 contributes to viral persistence in part by regulating host gene expression through binding host transcriptional regulators, although mechanisms responsible for E7-mediated transcriptional regulation are incompletely understood. Type I IFN signaling promotes the expression of anti-viral genes, called interferon-stimulated genes (ISGs), through the phosphorylation and activation of STAT1. In this study, we have observed that the CR3 domain of E7 contributes to the episomal maintenance of viral genomes. Transcriptome analysis revealed that E7 transcriptionally suppresses a subset of ISGs but not through regulation of STAT1 activation. Instead, we discovered that E7 associates with Mediator kinase CDK8 and this is correlated with the recruitment of CDK8 to ISG promoters and reduced ISG expression. E7 fails to suppress ISGs in the absence of CDK8, indicating that CDK8 function contributes to the suppression of ISGs by E7. Altogether, E7/CDK8 association may be a novel mechanism by which E7 inhibits innate immune signaling.

Human Papillomavirus 16 E5 Inhibits Interferon Signaling and Supports Episomal Viral Maintenance.

Human papillomaviruses (HPVs) infect keratinocytes of stratified epithelia. Long-term persistence of infection is a critical risk factor for the development of HPV-induced malignancies. Through the actions of its oncogenes, HPV evades host immune responses to facilitate its productive life cycle. In this work, we discovered a previously unknown function of the HPV16 E5 oncoprotein in the suppression of interferon (IFN) responses. This suppression is focused on keratinocyte-specific IFN-κ and is mediated through E5-induced changes in growth factor signaling pathways, as identified through phosphoproteomics analysis. The loss of E5 in keratinocytes maintaining the complete HPV16 genome results in the derepression of IFNK transcription and subsequent JAK/STAT-dependent upregulation of several IFN-stimulated genes (ISGs) at both the mRNA and protein levels. We also established a link between the loss of E5 and the subsequent loss of genome maintenance and stability, resulting in increased genome integration.IMPORTANCE Persistent human papillomavirus infections can cause a variety of significant cancers. The ability of HPV to persist depends on evasion of the host immune system. In this study, we show that the HPV16 E5 protein can suppress an important aspect of the host immune response. In addition, we find that the E5 protein is important for helping the virus avoid integration into the host genome, which is a frequent step along the pathway to cancer development.

Suppression of Stromal Interferon Signaling by Human Papillomavirus 16.

Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.

Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins.

BACKGROUND: Tospoviruses (genus Tospovirus, family Peribunyaviridae, order Bunyavirales) cause significant losses to a wide range of agronomic and horticultural crops worldwide. Identification and characterization of specific sequences and motifs that are critical for virus infection and pathogenicity could provide useful insights and targets for engineering virus resistance that is potentially both broad spectrum and durable. Tomato spotted wilt virus (TSWV), the most prolific member of the group, was used to better understand the structure-function relationships of the nucleocapsid gene (N), and the silencing suppressor gene (NSs), coded by the TSWV small RNA. METHODS: Using a global collection of orthotospoviral sequences, several amino acids that were conserved across the genus and the potential location of these conserved amino acid motifs in these proteins was determined. We used state of the art 3D modeling algorithms, MULTICOM-CLUSTER, MULTICOM-CONSTRUCT, MULTICOM-NOVEL, I-TASSER, ROSETTA and CONFOLD to predict the secondary and tertiary structures of the N and the NSs proteins. RESULTS: We identified nine amino acid residues in the N protein among 31 known tospoviral species, and ten amino acid residues in NSs protein among 27 tospoviral species that were conserved across the genus. For the N protein, all three algorithms gave nearly identical tertiary models. While the conserved residues were distributed throughout the protein on a linear scale, at the tertiary level, three residues were consistently located in the coil in all the models. For NSs protein models, there was no agreement among the three algorithms. However, with respect to the localization of the conserved motifs, G18 was consistently located in coil, while H115 was localized in the coil in three models. CONCLUSIONS: This is the first report of predicting the 3D structure of any tospoviral NSs protein and revealed a consistent location for two of the ten conserved residues. The modelers used gave accurate prediction for N protein allowing the localization of the conserved residues. Results form the basis for further work on the structure-function relationships of tospoviral proteins and could be useful in developing novel virus control strategies targeting the conserved residues.

Induction of Interferon Kappa in Human Papillomavirus 16 Infection by Transforming Growth Factor Beta-Induced Promoter Demethylation.

Persistent high-risk human papillomavirus (HPV) infection is the major causal factor in cervical and other anogenital cancers. Because there are currently no therapeutics capable of preventing neoplastic progression of HPV infections, understanding the mechanisms of HPV-mediated persistence, including immune evasion, is a major research priority. The multifunctional growth factor transforming growth factor beta (TGFß) has been shown to inhibit expression of early viral transcripts from cells harboring integrated HPV genomes or cells infected with retroviruses expressing HPV oncoproteins. However, the mechanism of TGFß-induced inhibition has not been fully defined. In this study, we have observed a previously uncharacterized ability of TGFß to repress the differentiation-induced upregulation of late HPV16 gene expression. In addition, interferon kappa (IFN-κ), a keratinocyte-specific, constitutively expressed cytokine suppressed by differentiation, can be transcriptionally induced by TGFß1. TGFß-mediated IFN-κ transcription only occurs in cells containing HPV16, and this is due to TGFß1-mediated reversal of HPV-induced methylation of the IFN-κ promoter through active DNA demethylation mediated by thymine DNA glycosylase (TDG). This novel interaction between growth factor and innate immune signaling may shed light on the mechanisms of HPV persistence and how the virus manipulates both immune and growth factor signaling to promote its life cycle.IMPORTANCE Persistent infection by high-risk HPVs is the primary risk factor for development of HPV-induced cancers. Persistence involves viral evasion of the immune response, including the IFN response. HPV is also known to suppress TGFß signaling, which inhibits viral gene expression. Here, we show that the TGFß and IFN pathways are interrelated in the context of HPV16 infection through the upregulation of IFN-κ by TGFß. The ability of TGFß to induce IFN-κ promoter demethylation and transcriptional activation provides a new explanation for why HPV has evolved mechanisms to inhibit TGFß in infected cells.

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins.

BACKGROUND: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and ß-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. CONCLUSION/SIGNIFICANCE: Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.

Movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections.

Negative-stranded tospoviruses (family: Bunyaviridae) are among the most agronomically important viruses. Some of the tospoviruses are known to exist as mixed infections in the same host plant. Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV) were used to study virus-virus interaction in dually infected host plants. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors. BiFC results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in dually infected Nicotiana benthamiana plants. BiFC results were further confirmed by pull down and yeast-2-hybrid (Y2H) assays. Interacting regions of the N and NSm proteins were also identified by Y2H system and ß-galactosidase activity. Several regions of the N and NSm were found interacting with each other. The regions involved in these interactions are presumed to be critical for the functioning of the tospovirus N and NSm proteins. This is the first report of in vivo protein interactions of distinct tospoviruses in mixed infection.

Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product.

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral infection. In order to perform its various tasks, P6 interacts with both viral and host factors, as well as forming electron-dense cytoplasmic inclusion bodies. Here we investigate the interactions of P6 with three CaMV proteins: P2 (aphid transmission factor), P3 (virion-associated protein), and P7 (protein of unknown function). Based on yeast two-hybrid and maltose-binding protein pull-down experiments, P6 interacted with all three of these CaMV proteins. P2 helps to stabilize P6 inclusion bodies. Although the P2s from two CaMV isolates (W260 and CM1841) differ in the ability to stabilize inclusion bodies, both interacted similarly with P6. This suggests that inclusion body stability may not be dependent on the efficiency of P2-P6 interaction. However, neither P2 nor P3 interacted with P7 in yeast two-hybrid assays.

The Dahlia mosaic virus gene VI product N-terminal region is involved in self-association.

The genome of the floriculture pathogen Dahlia mosaic caulimovirus (DMV) encodes six open reading frames. Generally, caulimovirus gene VI products (P6s) are thought to be multifunctional proteins required for viral infection and it is likely that self-association is required for some of these functions. In this study, yeast two-hybrid and maltose binding protein (MBP) pull-down assays indicated that full-length DMV P6 specifically self-associates. Further analyses indicated that only the DMV P6 N-terminal region, consisting of 115 amino acids, interacts with full-length P6 and with itself. This distinguishes the DMV P6 from its Cauliflower mosaic virus counterpart, which contains four regions involved in self-association. Thus, our results suggest that each caulimovirus P6 may possess a unique pattern of protein-protein interactions. Bioinformatic tools identified a putative nuclear exclusion signal located between amino acid residues 10-20, suggesting another possible function for the P6 N-terminal region.

Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein.

Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher beta-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1-P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.

Plant parasitic and vector nematodes associated with Asiatic and Oriental hybrid lilies.

A survey for distribution and abundance of plant parasitic nematodes in fields grown to Lilium in Himachal Pradesh, India at four study sites viz. Nagrota (at 810 m a.s.l.), Palampur (at 1270 m a.s.l.), Sunder Nagar (at 1400 m a.s.l.) and Chail (at 2250 m a.s.l.) was carried out. Moderate (101-500/200 ml soil) to high (501-1000/200 ml soil) populations of phytonematodes including the vectors for plant viruses (Aphelenchoides avenae, Criconemoides spp., Hoplolaimus spp., Longidorus spp., Paratylenchus spp., Pratylenchus spp., Rhabditis spp., Trichodorus spp., Tylenchoryhnchus spp., Tylenchulus spp. and Xiphinema diversicaudatum) were recorded. Mean population of nematodes was positively correlated with pH in all the study sites, negatively correlated with electrical conductivity (EC), percent organic matter (OM%), available potassium (K) and positively correlated with percent carbon (C%), available nitrogen (N) and available phosphorus (P) in all but one study site. The highest incidence of virus-vector nematodes viz. X. diversicaudatum, Longidorus spp. and Trichodorus spp. was recorded at Palampur. Only Strawberry latent ringspot nepovirus (SLRSV) was detected in Lilium cvs. Star Gazer Max and Galeili by Enzyme Linked Immunosorbent Assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) and in X. diversicaudatum associated with the cultivars by RT-PCR. Cucumis sativus used as bait plants showed SLRSV symptoms after 15 days of nematode inoculation and further SLRSV was again detected by ELISA and RT-PCR in C. sativus plants confirming the transmission of SLRSV by X. diversicaudatum in Lilium.