Puzzling functions of HSV-1 miRNAs in productive and latent infection

Herpes simplex virus (HSV) is a widespread human pathogen able to cause a broad spectrum of diseases of varying severity. HSV-1, and closely related virus HSV-2, employ a number of functions to evade host defense mechanisms and tailor the cellular environment during their complex life cycle comprised of productive and life-long latent infection. Once the latency is established, the virus can periodically reactivate in response to different stimuli and cause recurrent disease. Despite being one of the most intensively studied viruses, many biological aspects involved in the control of the lytic-latent switch and regulation of viral and host gene expression remained unclear. Discovery of the HSV-encoded miRNAs, a class of small regulatory RNAs, led to the hypothesis that they could have a central role in the establishment and maintenance of latency. HSV-1 and HSV-2 encode many miRNAs, differentially expressed during both phases of infection. The functions of HSV-encoded miRNAs have been experimentally addressed by several laboratories; however, the exact roles remain inconclusive. In this review, we will discuss the function of HSV-encoded miRNAs described to date, in terms of their viral and host targets, and the potential significance of such regulation for viral infection.</p

Analiza miRNA domaćina i herpes simpleks virusa 1 tijekom produktivne i latentne infekcije

Viruses significantly perturb cell metabolism during infection and guide their functions toward efficient virus replication. Previous studies have shown that herpes simplex virus 1, while also encoding its own microRNAs (miRNAs), leads to significant changes in host miRNA expression. However, the exact role of such deregulation has not been elucidated. In this study, we comprehensively analyzed host and HSV-1 encoded miRNAs during productive and latent infection by applying a next-generation sequencing approach and bioinformatic analyses followed by functional studies. Firstly, to study host miRNA changes during productive HSV-1 infection, we determined differentially expressed miRNAs in various HSV-1 infected cells in culture. We found a cluster of co-expressed host miRNAs, miR-183/96/182, that was reproducibly upregulated in primary cells. Interestingly, we found that miRNAs of this cluster share common targets, including the Forkhead box O (FoxO) family of transcription factors. We observed a slight increase followed by a decrease of FoxO1 and FoxO3 transcripts and proteins during HSV-1 infection, which coincided with the expression of the miR-183/96/182. However, we found that overexpression of the miR-183/96/182 cluster is not required for the depletion of FoxO proteins. Nonetheless, to further examine the roles of these proteins in infection, we generated cells deficient for FoxO1 and FoxO3 using the CRISPRCas9 technology. Our results show that individual FoxO1 or FoxO3 protein is not required for efficient virus infection. In the second part of the study, we analyzed HSV-1 miRNAs in great detail by comparing a large number of sequencing experiments (i.e., datasets). One part of the datasets was generated in-house by sequencing, and the other was obtained from public databases. Thus far, 29 mature miRNAs have been found encoded by HSV-1, the functions of which are largely unknown. However, we have noticed significant discrepancies between reports including a lack of consistency in miRNA sequences.Tijekom infekcije virusi značajno mijenjaju biologiju stanice, te pritom podređuju i usmjeravaju njezine funkcije u smjeru efikasne virusne replikacije. Prethodne studije su pokazale da herpes simpleks virus 1, osim što kodira svoje mikroRNA (miRNA), dovodi i do značajnih promjena u ekspresiji miRNA domaćina, za što se smatra da bi moglo dovoditi do stvaranja pogodnih uvjeta za replikaciju virusa, no točne uloge tih promjena nisu detaljno analizirane. Kako bi opširnije prikazali analizu miRNA domaćina te HSV-1, istraživanje je podijeljeno na dva dijela, prvi dio, koji čini analiza diferencijalnih promjena miRNA domaćina tijekom infekcije te drugi dio gdje su detaljnije analizirane miRNA HSV-1. Prvo, kako bi proučili promjene miRNA domaćina tijekom infekcije HSV-1, napravili smo profil diferencijalno eksprimiranih miRNA, te pokazali da je klaster miRNA domaćina koje se zajedno eksprimiraju, miR-183/96/182, reproducibilno povećano eksprimiran u primarnim stanicama u kulturi. Traženjem zajedničkih meta navedenih miRNA, od potencijalnih kandidata izdvojili smo Forkhead box O (FoxO) porodicu transkripcijskih faktora te uočili njihovo blago povećanje nakon kojeg slijedi opadanje razine transkripata te proteina tokom infekcije HSV-1. Kako bi ispitali ulogu FoxO u infekciji, metodom CRISPR-Cas9 generirali smo stanice deficijentne za FoxO1 i FoxO3, no, uspoređujući replikaciju, uočili smo da FoxO1 ili FoxO3 ne igraju značajnu ulogu u replikaciji HSV-1. Drugo, kako bi detaljnije analizirali miRNA HSV-1, generirali smo i prikupili setove podataka dobivene sekvenciranjem malih RNA molekula uzoraka inficiranih sa HSV-1. Istraživanja su do sada pokazala da HSV-1 kodira 29 zrelih miRNA, čija funkcija nije u potpunosti razjašnjena. U tim dosadašnjim otkrićima sudjelovale su različite istraživačke grupe koje su u svojim istraživanjima primjenjivale različite kriterije za otkrića novih miRNA što je na kraju rezultiralo brojnim odstupanjima, uključujući i točne sekvence miRNA HSV-1. Kako bismo odredili točne sekvence miRNA HSV-1, te ispitali ekspresiju miRNA u latenciji u uzorcima čovjeka, usporedili smo sekvence miRNA između različitih virusnih sojeva i kliničkih izolata. Opsežna bioinformatička analiza pokazala je odstupanja sekvenci miRNA HSV-1 od objavljenih referentnih sekvenci, čime smo definirali najdominantnije sekvence 29 zrelih miRNA molekula HSV-1 u različitim fazama HSV-1 infekcije kako bi doprinijeli razumijevanju i olakšali buduće funkcionalne analize ovih miRNA molekula. Također smo kategorizirali sve poznate miRNA HSV-1 te sugeriramo da neke od prije objavljenih miRNA možda nisu izvorne regulatorne molekule, već artefakti sekvenciranja. Nadalje, opisujemo značajno posttranskripcijsko uređivanje hsv1-miR-H2-3p u latentno inficiranim ganglijima čovjeka, za razliku od in vitro uzoraka u produktivnoj fazi infekcije. Ovi rezultati ukazuju na to da virus koristi stanične procese kako bi proširio repertoar mogućih meta miRNA ili na taj način utječe na njihovu stabilnost

Analiza miRNA domaćina i herpes simpleks virusa 1 tijekom produktivne i latentne infekcije

Viruses significantly perturb cell metabolism during infection and guide their functions toward efficient virus replication. Previous studies have shown that herpes simplex virus 1, while also encoding its own microRNAs (miRNAs), leads to significant changes in host miRNA expression. However, the exact role of such deregulation has not been elucidated. In this study, we comprehensively analyzed host and HSV-1 encoded miRNAs during productive and latent infection by applying a next-generation sequencing approach and bioinformatic analyses followed by functional studies. Firstly, to study host miRNA changes during productive HSV-1 infection, we determined differentially expressed miRNAs in various HSV-1 infected cells in culture. We found a cluster of co-expressed host miRNAs, miR-183/96/182, that was reproducibly upregulated in primary cells. Interestingly, we found that miRNAs of this cluster share common targets, including the Forkhead box O (FoxO) family of transcription factors. We observed a slight increase followed by a decrease of FoxO1 and FoxO3 transcripts and proteins during HSV-1 infection, which coincided with the expression of the miR-183/96/182. However, we found that overexpression of the miR-183/96/182 cluster is not required for the depletion of FoxO proteins. Nonetheless, to further examine the roles of these proteins in infection, we generated cells deficient for FoxO1 and FoxO3 using the CRISPRCas9 technology. Our results show that individual FoxO1 or FoxO3 protein is not required for efficient virus infection. In the second part of the study, we analyzed HSV-1 miRNAs in great detail by comparing a large number of sequencing experiments (i.e., datasets). One part of the datasets was generated in-house by sequencing, and the other was obtained from public databases. Thus far, 29 mature miRNAs have been found encoded by HSV-1, the functions of which are largely unknown. However, we have noticed significant discrepancies between reports including a lack of consistency in miRNA sequences.Tijekom infekcije virusi značajno mijenjaju biologiju stanice, te pritom podređuju i usmjeravaju njezine funkcije u smjeru efikasne virusne replikacije. Prethodne studije su pokazale da herpes simpleks virus 1, osim što kodira svoje mikroRNA (miRNA), dovodi i do značajnih promjena u ekspresiji miRNA domaćina, za što se smatra da bi moglo dovoditi do stvaranja pogodnih uvjeta za replikaciju virusa, no točne uloge tih promjena nisu detaljno analizirane. Kako bi opširnije prikazali analizu miRNA domaćina te HSV-1, istraživanje je podijeljeno na dva dijela, prvi dio, koji čini analiza diferencijalnih promjena miRNA domaćina tijekom infekcije te drugi dio gdje su detaljnije analizirane miRNA HSV-1. Prvo, kako bi proučili promjene miRNA domaćina tijekom infekcije HSV-1, napravili smo profil diferencijalno eksprimiranih miRNA, te pokazali da je klaster miRNA domaćina koje se zajedno eksprimiraju, miR-183/96/182, reproducibilno povećano eksprimiran u primarnim stanicama u kulturi. Traženjem zajedničkih meta navedenih miRNA, od potencijalnih kandidata izdvojili smo Forkhead box O (FoxO) porodicu transkripcijskih faktora te uočili njihovo blago povećanje nakon kojeg slijedi opadanje razine transkripata te proteina tokom infekcije HSV-1. Kako bi ispitali ulogu FoxO u infekciji, metodom CRISPR-Cas9 generirali smo stanice deficijentne za FoxO1 i FoxO3, no, uspoređujući replikaciju, uočili smo da FoxO1 ili FoxO3 ne igraju značajnu ulogu u replikaciji HSV-1. Drugo, kako bi detaljnije analizirali miRNA HSV-1, generirali smo i prikupili setove podataka dobivene sekvenciranjem malih RNA molekula uzoraka inficiranih sa HSV-1. Istraživanja su do sada pokazala da HSV-1 kodira 29 zrelih miRNA, čija funkcija nije u potpunosti razjašnjena. U tim dosadašnjim otkrićima sudjelovale su različite istraživačke grupe koje su u svojim istraživanjima primjenjivale različite kriterije za otkrića novih miRNA što je na kraju rezultiralo brojnim odstupanjima, uključujući i točne sekvence miRNA HSV-1. Kako bismo odredili točne sekvence miRNA HSV-1, te ispitali ekspresiju miRNA u latenciji u uzorcima čovjeka, usporedili smo sekvence miRNA između različitih virusnih sojeva i kliničkih izolata. Opsežna bioinformatička analiza pokazala je odstupanja sekvenci miRNA HSV-1 od objavljenih referentnih sekvenci, čime smo definirali najdominantnije sekvence 29 zrelih miRNA molekula HSV-1 u različitim fazama HSV-1 infekcije kako bi doprinijeli razumijevanju i olakšali buduće funkcionalne analize ovih miRNA molekula. Također smo kategorizirali sve poznate miRNA HSV-1 te sugeriramo da neke od prije objavljenih miRNA možda nisu izvorne regulatorne molekule, već artefakti sekvenciranja. Nadalje, opisujemo značajno posttranskripcijsko uređivanje hsv1-miR-H2-3p u latentno inficiranim ganglijima čovjeka, za razliku od in vitro uzoraka u produktivnoj fazi infekcije. Ovi rezultati ukazuju na to da virus koristi stanične procese kako bi proširio repertoar mogućih meta miRNA ili na taj način utječe na njihovu stabilnost

Analiza miRNA domaćina i herpes simpleks virusa 1 tijekom produktivne i latentne infekcije

Viruses significantly perturb cell metabolism during infection and guide their functions toward efficient virus replication. Previous studies have shown that herpes simplex virus 1, while also encoding its own microRNAs (miRNAs), leads to significant changes in host miRNA expression. However, the exact role of such deregulation has not been elucidated. In this study, we comprehensively analyzed host and HSV-1 encoded miRNAs during productive and latent infection by applying a next-generation sequencing approach and bioinformatic analyses followed by functional studies. Firstly, to study host miRNA changes during productive HSV-1 infection, we determined differentially expressed miRNAs in various HSV-1 infected cells in culture. We found a cluster of co-expressed host miRNAs, miR-183/96/182, that was reproducibly upregulated in primary cells. Interestingly, we found that miRNAs of this cluster share common targets, including the Forkhead box O (FoxO) family of transcription factors. We observed a slight increase followed by a decrease of FoxO1 and FoxO3 transcripts and proteins during HSV-1 infection, which coincided with the expression of the miR-183/96/182. However, we found that overexpression of the miR-183/96/182 cluster is not required for the depletion of FoxO proteins. Nonetheless, to further examine the roles of these proteins in infection, we generated cells deficient for FoxO1 and FoxO3 using the CRISPRCas9 technology. Our results show that individual FoxO1 or FoxO3 protein is not required for efficient virus infection. In the second part of the study, we analyzed HSV-1 miRNAs in great detail by comparing a large number of sequencing experiments (i.e., datasets). One part of the datasets was generated in-house by sequencing, and the other was obtained from public databases. Thus far, 29 mature miRNAs have been found encoded by HSV-1, the functions of which are largely unknown. However, we have noticed significant discrepancies between reports including a lack of consistency in miRNA sequences.Tijekom infekcije virusi značajno mijenjaju biologiju stanice, te pritom podređuju i usmjeravaju njezine funkcije u smjeru efikasne virusne replikacije. Prethodne studije su pokazale da herpes simpleks virus 1, osim što kodira svoje mikroRNA (miRNA), dovodi i do značajnih promjena u ekspresiji miRNA domaćina, za što se smatra da bi moglo dovoditi do stvaranja pogodnih uvjeta za replikaciju virusa, no točne uloge tih promjena nisu detaljno analizirane. Kako bi opširnije prikazali analizu miRNA domaćina te HSV-1, istraživanje je podijeljeno na dva dijela, prvi dio, koji čini analiza diferencijalnih promjena miRNA domaćina tijekom infekcije te drugi dio gdje su detaljnije analizirane miRNA HSV-1. Prvo, kako bi proučili promjene miRNA domaćina tijekom infekcije HSV-1, napravili smo profil diferencijalno eksprimiranih miRNA, te pokazali da je klaster miRNA domaćina koje se zajedno eksprimiraju, miR-183/96/182, reproducibilno povećano eksprimiran u primarnim stanicama u kulturi. Traženjem zajedničkih meta navedenih miRNA, od potencijalnih kandidata izdvojili smo Forkhead box O (FoxO) porodicu transkripcijskih faktora te uočili njihovo blago povećanje nakon kojeg slijedi opadanje razine transkripata te proteina tokom infekcije HSV-1. Kako bi ispitali ulogu FoxO u infekciji, metodom CRISPR-Cas9 generirali smo stanice deficijentne za FoxO1 i FoxO3, no, uspoređujući replikaciju, uočili smo da FoxO1 ili FoxO3 ne igraju značajnu ulogu u replikaciji HSV-1. Drugo, kako bi detaljnije analizirali miRNA HSV-1, generirali smo i prikupili setove podataka dobivene sekvenciranjem malih RNA molekula uzoraka inficiranih sa HSV-1. Istraživanja su do sada pokazala da HSV-1 kodira 29 zrelih miRNA, čija funkcija nije u potpunosti razjašnjena. U tim dosadašnjim otkrićima sudjelovale su različite istraživačke grupe koje su u svojim istraživanjima primjenjivale različite kriterije za otkrića novih miRNA što je na kraju rezultiralo brojnim odstupanjima, uključujući i točne sekvence miRNA HSV-1. Kako bismo odredili točne sekvence miRNA HSV-1, te ispitali ekspresiju miRNA u latenciji u uzorcima čovjeka, usporedili smo sekvence miRNA između različitih virusnih sojeva i kliničkih izolata. Opsežna bioinformatička analiza pokazala je odstupanja sekvenci miRNA HSV-1 od objavljenih referentnih sekvenci, čime smo definirali najdominantnije sekvence 29 zrelih miRNA molekula HSV-1 u različitim fazama HSV-1 infekcije kako bi doprinijeli razumijevanju i olakšali buduće funkcionalne analize ovih miRNA molekula. Također smo kategorizirali sve poznate miRNA HSV-1 te sugeriramo da neke od prije objavljenih miRNA možda nisu izvorne regulatorne molekule, već artefakti sekvenciranja. Nadalje, opisujemo značajno posttranskripcijsko uređivanje hsv1-miR-H2-3p u latentno inficiranim ganglijima čovjeka, za razliku od in vitro uzoraka u produktivnoj fazi infekcije. Ovi rezultati ukazuju na to da virus koristi stanične procese kako bi proširio repertoar mogućih meta miRNA ili na taj način utječe na njihovu stabilnost

Herpes Simplex Virus 1 Deregulation of Host MicroRNAs

Viruses utilize microRNAs (miRNAs) in a vast variety of possible interactions and mechanisms, apparently far beyond the classical understanding of gene repression in humans. Likewise, herpes simplex virus 1 (HSV-1) expresses numerous miRNAs and deregulates the expression of host miRNAs. Several HSV-1 miRNAs are abundantly expressed in latency, some of which are encoded antisense to transcripts of important productive infection genes, indicating their roles in repressing the productive cycle and/or in maintenance/reactivation from latency. In addition, HSV-1 also exploits host miRNAs to advance its replication or repress its genes to facilitate latency. Here, we discuss what is known about the functional interplay between HSV-1 and the host miRNA machinery, potential targets, and the molecular mechanisms leading to an efficient virus replication and spread

Herpes simplex virus 1 miRNA sequence variations in latently infected human trigeminal ganglia

Human herpes simplex virus 1 (HSV-1) expresses numerous miRNAs, the function of which is not well understood. Several qualitative and quantitative analyses of HSV-1 miRNAs have been performed on infected cells in culture and animal models, however, there is very limited knowledge of their expression in human samples. We sequenced small-RNA libraries of RNA derived from human trigeminal ganglia latently infected with HSV-1 and Varicella zoster virus (VZV) and detected only a small subset of HSV-1 miRNA. The most abundantly expressed miRNAs are miR- H2, miRNA that regulates the expression of immediate early gene ICP0, and miR-H3 and -H4, both miRNAs expressed antisense to the transcript encoding the major neurovirulence factor ICP34.5. The sequence of many HSV-1 miRNAs detected in human samples was different from the sequences deposited in miRBase, which might significantly affect targeted functional analyses

HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia

Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus

The Virus-Induced Upregulation of the miR-183/96/182 Cluster and the FoxO Family Protein Members Are Not Required for Efficient Replication of HSV-1

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus