The Role of Novel Gene UL79 Encoded by Human Cytomegalovirus in Viral Replication

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world\u27s population. Even though HCMV infection is usually asymptomatic, it acts as an opportunistic pathogen and is the major cause of morbidity and mortality in immunocompromised individuals, including transplant recipients and AIDS/HIV patients. Understanding HCMV biology is critical to the development of HCMV therapeutics. However, our current knowledge of HCMV biology is limited by the fact that only less than half of the HCMV genes have been characterized, especially viral essential and augmenting genes which play critical roles in viral replication. To study the function of HCMV essential and augmenting genes, we devised a conditional approach to facilitate the analysis. In this approach, we constructed recombinant virus where the viral open reading frame (ORF) of interest is tagged with the destabilization domain FKBP (ddFKBP) which targets the fusion protein for rapid degradation. However, the fusion protein can be stabilized by the synthetic ligand Shield-1 (Shld-1). This system allows us to monitor the effects of the viral protein on the HCMV life cycle simply by infecting human fibroblasts in the presence or absence of Shld-1. We adopted this conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADddUL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with ddFKBP. ADddUL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early- late kinetics, and was also regulated via a positive-feedback loop. We also found that the UL79 protein localized to viral replication compartments during HCMV infection. Moreover, we created a second UL79 mutant virus (ADinUL79stop) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADinUL79stop phenocopied ADddUL79 under the destabilizing condition. Taking together, we showed that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts. To test how pUL79 regulates viral late gene transcription, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87, pUL92, and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. We showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which is critical for transcriptional regulation. Rather, nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on viral DNA. Surprisingly, pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79 regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection. Together these data provide important insight into how HCMV uses pUL79 to promote viral transcription specifically during late stages of viral infection. Understanding the mechanisms by which pUL79 regulates RNAP II elongation as well as its association with other viral factors will aid in the development of future therapeutics against HCMV infection

Human cytomegalovirus pUL79 Is an elongation factor of RNA polymerase II for viral gene transcription

In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection

ISG15 connects autophagy and IFN-γ-dependent control of Toxoplasma gondii infection in human cells

The intracellular protozoan parasit

Coexistent squamous cell carcinoma and adenoid basal carcinoma in the uterine cervix and infection with human papillomavirus (HPV 31)

AbstractObjectiveAdenoid basal carcinoma (ABC) is an uncommon neoplasm of the uterine cervix. ABC can be accompanied by carcinoma in situ or invasive carcinoma. Most cases are discovered accidentally during radical hysterectomy. ABC is associated with a high risk of human papillomavirus infection (HPV), most often HPV 16 infection.Case reportWe present a rare case of an 86-year-old Taiwanese married woman who suffered from bloody vaginal discharge and occasional lower abdominal pain and received cervical biopsy. The pathological report revealed squamous cell carcinoma (SCC) of the uterine cervix. After radical hysterectomy, bilateral salpingo-oophorectomy, and bilateral pelvic and para-aortic lymph node dissection, the final pathological report revealed SCC coexisting with ABC, and both of the components were infected by HPV 31. After receiving radiotherapy, she maintained outpatient department follow-up.ConclusionA literature review revealed that this was a rare case of combined ABC–SCC associated with HPV 31 infection. In this case, the ABC component did not affect the tumor stage because it was confined to the cervix. However, we must avoid overestimating the clinical stage because the ABC component is thought to be a benign lesion

6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro

The emergence of SARS-CoV-2 has led to a global health crisis that, in addition to vaccines and immunomodulatory therapies, calls for the identification of antiviral therapeutics. The papain-like protease (PLpro) activity of nsp3 is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG), an orally available and widely available generic drug, inhibits SARS-CoV-2 replication in Vero-E6 cells with an EC50 of approximately 2 μM. 6-TG also inhibited PLpro-catalyzed polyprotein cleavage and de-ISGylation in cells and inhibited proteolytic activity of the purified PLpro domai

Вихретоковый анизотропный термоэлектрический первичный преобразователь лучистого потока

Представлена оригинальная конструкция первичного преобразователя лучистого потока, который может служить основой для создания приемника неселективного излучения с повышенной чувствительностью

The 5p15.33 Locus Is Associated with Risk of Lung Adenocarcinoma in Never-Smoking Females in Asia

Genome-wide association studies of lung cancer reported in populations of European background have identified three regions on chromosomes 5p15.33, 6p21.33, and 15q25 that have achieved genome-wide significance with p-values of 10−7 or lower. These studies have been performed primarily in cigarette smokers, raising the possibility that the observed associations could be related to tobacco use, lung carcinogenesis, or both. Since most women in Asia do not smoke, we conducted a genome-wide association study of lung adenocarcinoma in never-smoking females (584 cases, 585 controls) among Han Chinese in Taiwan and found that the most significant association was for rs2736100 on chromosome 5p15.33 (p = 1.30×10−11). This finding was independently replicated in seven studies from East Asia totaling 1,164 lung adenocarcinomas and 1,736 controls (p = 5.38×10−11). A pooled analysis achieved genome-wide significance for rs2736100. This SNP marker localizes to the CLPTM1L-TERT locus on chromosome 5p15.33 (p = 2.60×10−20, allelic risk = 1.54, 95% Confidence Interval (CI) 1.41–1.68). Risks for heterozygote and homozygote carriers of the minor allele were 1.62 (95% CI; 1.40–1.87), and 2.35 (95% CI: 1.95–2.83), respectively. In summary, our results show that genetic variation in the CLPTM1L-TERT locus of chromosome 5p15.33 is directly associated with the risk of lung cancer, most notably adenocarcinoma

Characterization of Papain-like Protease of SARS-Coronavirus

SARS-冠狀病毒(SARS-CoV)是造成2003年嚴重急性呼吸道症候群大規模流行的致病源，屬於冠狀病毒屬。冠狀病毒相較一般的RNA病毒，具有較長的基因序列，因此，冠狀病毒的複製以及基因調控的機制比起其他病毒，也顯得更為複雜。一般預期SARS病毒的基因表現，轉譯以及後轉譯調控也非常複雜，詳細的分子作用機轉，需要更進一步研究來加以闡明。來自於SARS-CoV polyprotein ORF1a 的蛋白酶PLpro 被預期會切割在ORF1a N端的三個切點上。ORF1a上另外一個蛋白酶3C-like (3CLpro)則負責作用在其他的11個切點。1a以及1ab經蛋白酶作用所產生的蛋白質產物，一般相信會組合形成依附在細胞內膜上形成replication complex，對SARS病毒的複製，病毒產物的產生，以及致病力扮演重要的角色。 我們利用in vitro以及in vivo酵素活性測定的實驗設計，來檢視蛋白酶PLpro 的活性。首先，將in vitro合成的SARS-CoV PLpro 以及預測具有酵素切點的蛋白質片段混合，來測試酵素的活性。在in vitro實驗中，我們發現PLpro蛋白酶展現出cis-cleavage的活性，但卻沒有trans-cleavage的活性。然而，相同的酵素片段，在in vivo的環境下卻展現出具有相當高的trans-cleavage活性。這樣的實驗結果顯示，某些細胞因子對於PLpro酵素的trans-cleavage活性，扮演著重要且關鍵的角色。此外，透過這次的研究，我們在酵素受質3(substrate 3)的片段，一段含nsp2/nsp3 PLpro 切點的蛋白質片段上，卻發現到一個性質未知的蛋白酶切點。相對於substrate 3所包含的PLpro 切點，這個性質未知的切點，在沒有PLpro 蛋白酶出現的條件下，仍然會被切割，顯示作用在該切點的酵素並非PLpro。我們利用serial deletion的實驗將切點所在位置的範圍，縮小到約莫SARS ORF1a 蛋白質的第950~960個氨基酸之間。這個新切點的酵素作用，不只出現在利用rabbit reticulocyte lysate所合成的蛋白質上，由wheat germ lysate所合成的蛋白質片段，也顯示出相同的結果。然而，如果將合成的蛋白質從lysate中純化出來，類似的酵素作用立刻消失。因此，我們認為，用來合成蛋白質的lysate當中，可能含有某些蛋白酶，可作用在這個新的蛋白酶切點上。SARS-Coronavirus (SARS-CoV) is the causative agent of the epidemic of the severe acute respiratory syndrome (SARS) in 2003. Coronaviruses are the exceptionally large RNA viruses and employ complex regulatory mechanisms to replicate and express their genomes. Gene expression of SARS-CoV is believed to involve complicated transcriptional, translational and post-translational regulatory mechanisms, the molecular details of which, however, remain to be determined. The PLpro encoded in the SARS-CoV polyprotein ORF1a is predicted to process the N-terminal three sites of ORF1a and 1ab, whereas the 3C-like (3CL) protease is responsible for the cleavage of the rest sites. The products derived from 1a and 1ab are believed to assemble into a membrane-associated viral replication complex, facilitating the replication and downstream viral biogenesis as well as pathogenesis. In this study, we determined the enzyme activity of SARS-CoV PLpro by in vitro and in vivo assay systems. When incubating the in vitro synthesized SARS-CoV PLpro with its predicted substrates, the enzyme exhibited cis-cleavage activity but no detectable trans-cleavage activity in vitro. However, the same enzymatic domain trans-cleaved the substrates efficiently in vivo. The results suggest that some cellular factors of human cells may be important and necessary for the trans-cleavage activity of PLpro. Here, we also identified a cryptic cleavage site within substrate 3, a fragment that contained the predicted nsp2/nsp3 cleavage site for PLpro. The cryptic site was cleaved even in the absence of PLpro, suggesting that it was not a substrate site for PLpro. Serial deletion analysis further established that the cryptic site was around residues 950~960 of SARS-CoV ORF1a. The cryptic processing of substrate 3 was observed not only when the protein was synthesized in rabbit reticulocyte lysate, but also when it was synthesized in wheat germ lysate. However, the cryptic processing was no longer observed when the synthesized substrate 3 was purified from the lysate. Therefore, we concluded that the proteases from the lysate may mediate this cleavage phenomenon.中文摘要 …………………………………………………………… I 英文摘要 …………………………………………………………… III 序論 ………………………………………………………………… 1 1.嚴重急性呼吸道症候群(SARS)之歷史 ……………………… 1 2.SARS冠狀病毒之基因體結構及種源(phylogenic)分類 …… 2 3. SARS冠狀病毒之複合蛋白質(polyprotein)及其複製 ……… 4 4. SARS病毒蛋白酶的重要性以及其藥理學上的含意 ……… 5 5. 本實驗的目的 …………………………………………………7 材料與方法 ………………………………………………………… 8 1.病毒，細胞與抗體 …………………………………………… 8 2. SARS PLpro基因片段與受質(substrates) 片段之質體構築… 9 3. 點突變 ……………………………………………………… 10 4. 試管內 (in vitro) 轉錄及轉譯所進行之trans-cleavage 分析…10 5. 利用痘病毒系統在HeLa細胞內(in vivo) 所進行之trans-cleavage 分析 …………………………………………………… 11 6. 利用痘病毒系統在293T細胞內表現酵素受質進行cleavage活性析 ………………………………………………………………12 7. 放射性免疫沈澱分析 ………………………………………… 12 8. 表現並純化His-標記之酵素受質3 ………………………… 13 結果 ………………………………………………………………… 15 1. 分析SARS-CoV 之PLpro 在試管中之cis-cleavage 活性… 15 2. 分析SARS-CoV 之PLpro 在試管中之trans-cleavage 活性 16 3. 分析SARS-CoV 之 PLpro 在細胞內之trans-cleavage 活性 19 4. 檢驗nsp 3 蛋白內異常切割的可能機制 …………………… 22 5. 利用系列刪除法來決定未知蛋白酶的切割點 …………… 23 6. 在不同表達系統中所合成之蛋白質依舊具有未知蛋白酶切割的現象 …………………………………………………………… 25 7. 純化過之合成受質3不再具有未知蛋白酶切割的情形 … 28 討論 ……………………………………………………………… 30 1.SARS-CoV 的 PLpro ……………………………………… 30 2.未知蛋白酶切割點的研究 ………………………………… 34 未來實驗方向與展望 …………………………………………… 37 參考文獻 ……………………………………………………… 38 圖與表 …………………………………………………………… 4