Levels Of Viral Gene Expression Research Articles

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Dynamic gene expression of southern rice black-streaked dwarf virus during its infection in rice plants and vector insects

In this century, a disease caused by southern rice black-streaked dwarf virus (SRBSDV) has resulted in significant loss in rice production in Asia. Aside from infecting rice plants, SRBSDV is transmitted by white-backed planthopper (WBPH) in a persistent propagative manner. Recent studies showed that SRBSDV can dynamically modulate the host cells throughout the infection progress. However, the expression dynamics of the SRBSDV genes during infection remain unclear. Here we established an absolute real-time quantitative PCR method to assess the dynamic of the SRBSDV genes expression in rice plants and planthoppers. Apart from displaying the expression levels of viral genes, we discovered that the expression level of viral genes in insects significantly surpasses that in plant cells. In addition, we identified two nonstructural proteins with unknown functions that exhibit the highest expression levels in plant and insect cells, respectively, which provide possible targets for restraining the disease outbreaks.

Cell-permeable peptide nucleic acid antisense oligonucleotide platform targeting human betacoronaviruses.

Antisense oligonucleotides (ASOs) with therapeutic potential have recently been reported to target the SARS-CoV-2 genome. Peptide nucleic acids (PNAs)-based ASOs have been regarded as promising drug candidates, but intracellular delivery has been a significant obstacle. Here, we present novel modified PNAs, termed OPNAs, with excellent cell permeability that disrupt the RNA genome of SARS-CoV-2 and HCoV-OC43 by introducing cationic lipid moiety onto the nucleobase of PNA oligomer backbone. HCT-8 cells and Caco-2 cells were treated with 1 μM antisense OPNAs at the time of viral challenge and the Viral RNA levels were measured by RT-qPCR three days post infection. NSP 14 targeting OPNA 5 and 11, reduced the viral titer to a half and OPNA 530, 531 and 533 lowered viral gene expression levels to less than 50% of control by targeting the 5' UTR region. Several modifications (oligo size and position, etc.) were introduced to enhance the efficacy of selected OPNAs. Improved OPNAs exhibited a dose-dependent reduction in viral replication and nucleoprotein (NP) protein. When a mixture of oligomers was applied to infected cells, viral titer and NP levels decreased by more than eightfold. In this study, we have developed a modified PNA ASO platform with exceptional chemical stability, high binding affinity, and cellular permeability. These findings indicate that OPNAs are a promising platform for the development of antivirals to combat future pandemic viral infections that do not require a carrier.

Multi-year molecular quantification and 'omics analysis of Planktothrix-specific cyanophage sequences from Sandusky Bay, Lake Erie.

Planktothrix agardhii is a microcystin-producing cyanobacterium found in Sandusky Bay, a shallow and turbid embayment of Lake Erie. Previous work in other systems has indicated that cyanophages are an important natural control factor of harmful algal blooms. Currently, there are few cyanophages that are known to infect P. agardhii, with the best-known being PaV-LD, a tail-less cyanophage isolated from Lake Donghu, China. Presented here is a molecular characterization of Planktothrix specific cyanophages in Sandusky Bay. Putative Planktothrix-specific viral sequences from metagenomic data from the bay in 2013, 2018, and 2019 were identified by two approaches: homology to known phage PaV-LD, or through matching CRISPR spacer sequences with Planktothrix host genomes. Several contigs were identified as having viral signatures, either related to PaV-LD or potentially novel sequences. Transcriptomic data from 2015, 2018, and 2019 were also employed for the further identification of cyanophages, as well as gene expression of select viral sequences. Finally, viral quantification was tested using qPCR in 2015-2019 for PaV-LD like cyanophages to identify the relationship between presence and gene expression of these cyanophages. Notably, while PaV-LD like cyanophages were in high abundance over the course of multiple years (qPCR), transcriptomic analysis revealed only low levels of viral gene expression. This work aims to provide a broader understanding of Planktothrix cyanophage diversity with the goals of teasing apart the role of cyanophages in the control and regulation of harmful algal blooms and designing monitoring methodology for potential toxin-releasing lysis events.

Molecular characterization of human cytomegalovirus infection with single-cell transcriptomics.

Human cytomegalovirus (HCMV) can result in either productive or non-productive infection, with the latter potentially leading to viral latency. The molecular factors dictating these outcomes are poorly understood. Here we used single-cell transcriptomics to analyse HCMV infection progression in monocytes, which are latently infected, and macrophages, considered to be permissive for productive infection. We show that early viral gene expression levels, specifically of those encoding immediate early proteins IE1 and IE2, are a major factor dictating productive infection. We also revealed that intrinsic, not induced, host cell interferon-stimulated gene expression level is a main determinant of infection outcome. Intrinsic interferon-stimulated gene expression is downregulated with monocyte to macrophage differentiation, partially explaining increased macrophage susceptibility to productive HCMV infection. Furthermore, non-productive macrophages could reactivate, making them potential latent virus reservoirs. Overall, we decipher molecular features underlying HCMV infection outcomes and propose macrophages as a potential HCMV reservoir.

Molecular cloning, expression and functional analysis of STAT2 in orange-spotted grouper, Epinephelus coioides

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

Comparison of viral distribution in duck hepatitis A virus-infected duckling models established by two different methods

Duck hepatitis A virus type 1 (DHAV-1) infection is the main cause of duck viral hepatitis, but the replication process and distribution of DHAV-1 in vivo are still poorly understood. In this study, six-day-old ducklings were infected by two different methods: by intramuscular injection to establish DHAV-1 infection animal models and by the combined administration of virus solution orally, through nasal inhalation, through inoculation of the eye, and through intrarectal contact to simulate natural infection. Tissues were collected at different time points and quantitative real-time polymerase chain reaction (qPCR) was employed to analyze the gene expression levels of DHAV-1 in different tissues. The results showed that the viral gene levels responded to the different challenge methods. Viral gene expression levels in all tissues in the intramuscular injection group were lower than those in the group that simulated natural infection. In both groups, the liver was the primary tissue that responsible for the replication of DHAV-1 genes, as virus gene level peaked at 4 h post infection (hpi). In addition, the respiratory and digestive tracts were important regions for DHAV-1 infection as high viral gene levels were detected at early (8 hpi) and late (96 hpi) stages of infection. This research utilized a novel infection method to simulate natural infection and analyzed the DHAV-1 distribution in different tissues. The findings can provide guidance for making prevention and control measures.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

Diversity in responses to oncolytic Lassa-vesicular stomatitis virus in patient-derived glioblastoma cells

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.

Palmitic Acid Promotes Virus Replication in Fish Cell by Modulating Autophagy Flux and TBK1-IRF3/7 Pathway.

Palmitic acid is the most common saturated fatty acid in animals, plants, and microorganisms. Studies highlighted that palmitic acid plays a significant role in diverse cellular processes and viral infections. Accumulation of palmitic acid was observed in fish cells (grouper spleen, GS) infected with Singapore grouper iridovirus (SGIV). The fluctuated content levels after viral infection suggested that palmitic acid was functional in virus-cell interactions. In order to investigate the roles of palmitic acid in SGIV infection, the effects of palmitic acid on SGIV induced cytopathic effect, expression levels of viral genes, viral proteins, as well as virus production were evaluated. The infection and replication of SGIV were increased after exogenous addition of palmitic acid but suppressed after knockdown of fatty acid synthase (FASN), of which the primary function was to catalyze palmitate synthesis. Besides, the promotion of virus replication was associated with the down-regulating of interferon-related molecules, and the reduction of IFN1 and ISRE promotor activities by palmitic acid. We also discovered that palmitic acid restricted TBK1, but not MDA5-induced interferon immune responses. On the other hand, palmitic acid decreased autophagy flux in GS cells via suppressing autophagic degradation, and subsequently enhanced viral replication. Together, our findings indicate that palmitic acid is not only a negative regulator of TBK1-IRF3/7 pathway, but also a suppressor of autophagic flux. Finally, palmitic acid promotes the replication of SGIV in fish cells.

HSV-1 single-cell analysis reveals the activation of anti-viral and developmental programs in distinct sub-populations.

Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of β-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance

Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance.

Transcriptomic profiles of striped snakehead cells (SSN-1) infected with snakehead vesiculovirus (SHVV) identifying IFI35 as a positive factor for SHVV replication

Snakehead vesiculovirus (SHVV) has caused great economic loss in snakehead fish culture in China. However, there is no effective strategy to prevent the epidemic of the virus. Understanding the host factors in response to virus infection is the basis for the prevention of viral disease. In this study, the transcriptomic profiles of SHVV-infected and mock-infected SSN-1 cells (derived from striped snakehead, Channa striatus) at 3 and 24 h (h) post of infection (poi) were obtained using high-throughput sequencing technique. A total of 93,372 unigenes were obtained. The differently expressed genes (DEGs) of SSN-1 cells upon SHVV infection were thereby identified, including 3668 and 3536 DEGs at 3 and 24 h poi, respectively. These DEGs were involved in many pathways of viral pathogenesis, including retinoic acid-inducible gene I (RIG-I) like receptors pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Therefore, several immune-related DEGs were randomly selected and confirmed by quantitative real-time PCR (qRT-PCR). In addition, the effects of the interferon inducible protein 35 (IFI35) on SHVV replication were further investigated. Over-expression or inhibition of IFI35 significantly promoted or reduced SHVV replication at the level of viral gene expression, which indicated that IFI35 might be a positive factor for SHVV replication in SSN-1 cells. Our findings presented some valuable information, which will benefit for future study on SHVV-host interactions.

Olea europaea leaves extract downregulate Newcastle disease virus gene expression in cancer cells

Many researches have been made to find antiviral agents from natural sources. Leaf of Olea europaea (OLE) is one of themost important medicinal plants, as it is a well-known source of different phytochemicals. This study was carried out toinvestigate the effect of using Olea europaea leaves extract on the level of viral gene expression (M and F) in Hela cell line byusing the qRT-PCR. Also, we examined the adding of interferon-beta ( IFN-b ) as an antivirus factor and analyzed the expressionof viral and IFN-b genes to identify viral targets for OLE extract. First, we propagated the virus in embryonated chickeneggs and showed to kill embryos in different times intervals (less than 72 hours) with marked sever hemorrhage in infectedembryos, and agglutination activity reflects titer (1024) and 1×10 6.4 (TCID50). Hela cells were cultured and different concentrationof OLE extract was added; cell viability was measured by MTT assay. Gene expression showed that the expression ofthe viral gene was downregulated in the present of OLE extract. Pretreatment with IFN-b downregulated viral gene expressionhigher than the extract, also evaluated the IFN-b concentration by ELISA assay. OLE can be considered as antivirus agent andused this property to help in control Newcastle disease.

Grouper STAT1a is involved in antiviral immune response against iridovirus and nodavirus infection

Signal Transducer and Activator of Transcription 1 (STAT1) has been demonstrated to function as a critical mediator in multiple cell processes, such as cell proliferation, cell death, and innate immune response. Interestingly, two orthologues of human STAT1, including STAT1a and STAT1b genes have been identified in different fish. However, the detailed roles of fish STAT1a in virus replication still remained largely uncertain. Here, we cloned a STAT1a from orange-spotted grouper Epinephelus coioides (EcSTAT1a) and characterized its roles during fish virus infection. EcSTAT1a encoded a 751-aa peptide which shared 97% and 93% identity to STAT1 from mandarin fish (Siniperca chuatsi) and Malabar grouper (Epinephelus malabaricus), respectively. Amino acid alignment analysis showed that EcSTAT1a contained a STAT-int domain, a STAT-alpha domain, a STAT-bind domain (DNA binding domain), a SH2 domain and a STAT1-TAZ2 bind domain. In examined tissues from healthy grouper, the expression of EcSTAT1a was predominant in intestine, gill and liver. In grouper cells, the relative expression levels of EcSTAT1a was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) or Singapore grouper iridovirus (SGIV) infection. Under fluorescence microscopy, we found that EcSTAT1a mainly localized in the cytoplasm. The ectopic expression of EcSTAT1a in vitro significantly delayed the cytopathic effect (CPE) progression evoked by RGNNV and SGIV. Further studies showed that the expression levels of viral genes, including SGIV major capsid protein (MCP), VP19, ICP-18, LITAF and RGNNV coat protein (CP), RNA-dependent RNA polymerase (RdRp) were all significantly reduced in EcSTAT1a overexpressing cells compared to the control vector transfected cells, suggested that EcSTAT1a exerted antiviral activity against iridovirus and nodavirus. Furthermore, overexpression of EcSTAT1a significantly increased the expression of interferon related cytokines or effectors and pro-inflammatory factors. Together, our results elucidated that EcSTAT1a might function as a critical antiviral factor by regulating the host interferon immune and inflammation response.

Co-infection of two reoviruses increases both viruses accumulation in rice by up-regulating of viroplasm components and movement proteins bilaterally and RNA silencing suppressor unilaterally

BackgroundSynergism between southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) not only aggravates disease symptoms but also enhances their vector acquisition efficiencies by increasing both viruses’ titers in co-infected rice plants, which may exacerbate the epidemic of both viruses and cause significant damage to rice production. The molecular mechanism of viral synergism of these two viruses remains unexplored.MethodsSingle and double infection of SRBSDV and RRSV were obtained with the viruliferous white-backed planthopper and brown planthopper inoculation on four-leaf stage rice seedlings, respectively, under experimental condition. The second upper leaf from each inoculated rice plants were collected at 9, 15, and 20 days post inoculation (dpi) and used for relative quantification of 13 SRBSDV genes and 11 RRSV genes by the reverse-transcription quantitative PCR. Viral gene expression levels were compared between singly and doubly infected samples at the same stage.ResultsThe movement protein and viroplasm matrix-related genes as well as the structural (capsid) protein genes of both viruses were remarkably up-regulated at different time points in the co-infected rice plants compared with the samples singly infected with SRBSDV or RRSV, however, the RNA silencing suppressor (P6) of only RRSV, but not of both the viruses, was up-regulated.ConclusionsThe SRBSDV-RRSV synergism promoted replication and movement of both viruses and inhibited the host immunity by enhancing the gene suppressing effect exerted by one of them (RRSV).

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study.

A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

PA-seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma-Associated Herpesvirus Transcripts.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differ from what is observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site). This represents a challenge in determination of the 3' end of individual viral transcripts. Such information is critical for generation of a virus transcriptional map for genetic studies. Here we present PA-seq, a high-throughput method for genome-wide analysis of pA sites of KSHV transcripts in B lymphocytes with latent or lytic KSHV infection. Besides identification of all viral pA sites, PA-seq also provides quantitative information about the levels of viral transcripts associated with each pA site, making it possible to determine the relative expression levels of viral genes at various stages of infection. Due to the indiscriminate nature of PA-seq, the pA sites of host transcripts are also concurrently mapped in the testing samples. Therefore, this technology can simultaneously estimate the expression changes of host genes and RNA polyadenylation upon KSHV infection. © 2016 by John Wiley & Sons, Inc.

KLF13 regulates the differentiation-dependent human papillomavirus life cycle in keratinocytes through STAT5 and IL-8.

High-risk strains of human papillomavirus (HPV) are the causative agents of cervical and anogenital cancers and are associated with 5% of all human cancers. Although prophylactic vaccines targeting a subset of HPV types are available, they are ineffective in HPV-infected individuals. Elucidation of the mechanisms controlling HPV replication may allow development of novel anti-HPV therapeutics. Infectious HPV virions are produced during terminal differentiation of host cells. The process of viral maturation requires synergistic interactions between viral and cellular proteins that leads to amplification of the viral genome and expression of late viral genes. Here we show that the transcription factor Kruppel-like factor 13 (KLF13) has a critical role in the HPV life cycle. KLF13 is overexpressed in HPV-positive keratinocytes and cervical cancer cell lines. Expression of KLF13 in normal cervical epithelium is low but increases significantly in cervical intraepithelial neoplasia and invasive squamous cervical cancer. After HPV infection, the E7 protein suppresses ubiquitin ligase FBW7 expression leading to an increase in KLF13 expression. Reduction of KLF13 with short hairpin RNA in differentiating HPV-positive cells resulted in diminished levels of viral gene expression and genome amplification. Knockdown of KLF13 also reduced the level of the transcription factor signal transducer and activator of transcription 5, which led to the downregulation of the ataxia-telangiectasia mutated DNA damage pathway and the chemokine interleukin-8 (IL-8). In addition, neutralization of IL-8 diminished viral genome amplification in differentiating HPV-positive cells. Thus, KLF13 is critical for the activation of the HPV productive life cycle and is likely involved in initiation and progression of cervical cancer.