­­Assessment of Viral RNA in Idiopathic Pulmonary Fibrosis Using RNA-seq

Background Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts. Methods We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 age-matched controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K). Results We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs. Conclusions To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

­­Assessment of Viral RNA in Idiopathic Pulmonary Fibrosis Using RNA-seq

Background Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts. Methods We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 age-matched controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K). Results We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs. Conclusions To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

Engineering a recombinant Herpesvirus saimiri strain by co-culturing transfected and permissive cells

Recombinant herpesviruses can be used as oncolytic therapeutic agents and high packaging capacity vectors for delivering expression cassettes into the cell. Herpesvirus saimiri is a gamma-herpesvirus that normally infects squirrel monkeys but also has a unique ability to infect and immortalize human lymphocytes while allowing them to retain their mature phenotype and functional activity. Recombination of the Herpesvirus saimiri genome in permissive cells is impeded by its resistance to chemical transfection and electroporation. The aim of this study was to develop an effective method for incorporating expression cassettes into the genome of Herpesvirus saimiri without having to transfect a permissive cell culture. Transfected HEK-293T cells expressing glycoproteins of the measles virus vaccine strain were co-cultured with permissive OMK cells infected with Herpesvirus saimiri. Cell fusion and formation of syncytia stimulated recombination between the viral genome and the expression cassette; this allowed us to obtain a recombinant Herpesvirus saimiri variant without chemical transfection in permissive cells. The genetically modified virus expressed a selectable marker and retained its ability to persist in the cell in the latent state; it also caused immortalization of primary lymphoid cells. The proposed approach allows engineering recombinant Herpesvirus saimiri strains carrying a variety of expression cassettes in its genome.

Получение рекомбинантного штамма Herpesvirus saimiri путем совместной культивации трансфицированной и пермиссивной клеточных культур

Рекомбинантные герпесвирусы можно применять в качестве терапевтических агентов-онколитиков, а также в качестве векторов большой емкости для доставки протяженных экспрессионных конструкций в клетки. Гамма-герпесвирус беличьих обезьян Herpesvirus saimiri обладает уникальной способностью инфицировать человеческие лимфоциты и вызывать их иммортализацию при сохранении зрелого фенотипа и функциональной активности. Проведение рекомбинации генома Herpesvirus saimiri в пермиссивной клеточной культуре затруднено из-за ее устойчивости к химической трансфекции и электропорации. Целью работы являлась разработка эффективного способа введения экспрессионных кассет в геном Herpesvirus saimiri без проведения трансфекции пермиссивной клеточной культуры. Для этого мы использовали совместную культивацию трансфицированных клеток HEK-293T, экспрессирующих также гликопротеины вакцинного штамма вируса кори, и инфицированных Herpesvirus saimiri пермиссивных клеток линии ОМК. Слияние клеток и образование синцитиев привели к запуску рекомбинации между вирусным геномом и экспрессионной кассетой, что позволило получить рекомбинантный вариант Herpesvirus saimiri без необходимости проведения химической трансфекции пермиссивных клеток. Трансгенный вариант вируса характеризовался стабильной экспрессией селективного маркера и сохранял способность персистировать в клеточной культуре в латентной форме, а также вызывать иммортализацию первичных лимфоидных клеток. Примененный метод позволяет в короткие сроки получать рекомбинантные варианты Herpesvirus saimiri с введенными в геном разнообразными экспрессионными кассетами.

­­Assessment of Viral RNA in Idiopathic Pulmonary Fibrosis Using RNA-seq

Background Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts. Methods We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 age-matched controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K). Results We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs. Conclusions To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

­­Assessment of Viral RNA in Idiopathic Pulmonary Fibrosis Using RNA-seq

Background Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts. Methods We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 age-matched controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K). Results We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs. Conclusions To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators

ABSTRACTIn latently infected marmoset T cells,Herpesvirus saimiri(HVS) expresses six microRNAs (known as miR-HSURs [H. saimiriU-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency.IMPORTANCEViruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.