المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: Immunoblot was performed using anti-LANA or anti-GFP antibody following infection of 293 cells with KSHV or KSHVΔLANA, or following transient tranfection of GFP LANA 779–1162 or GFP LANA 933–1162 in 293 cells. LANA, GFP, GFP LANA 779–1162 or GFP LANA 933–1162 bands are indicated by asterisks to the right of each band. GFP is expressed from the recombinant KSHV or KSHVΔLANA genomes. Control lane contains uninfected and untransfected 293 cells. GFP LANA 779–1162 contains LANA repeat elements, accounting for increased signal with the LANA polyclonal antibody compared to GFP LANA 933–1162, despite its lower expression, as observed in the anti-GFP immunoblot. (TIF)

العلاقة: https://figshare.com/articles/figure/KSHV_LANA_does_not_express_truncated_LANA_products_from_downstream_initiation_of_translation_/25015285Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.s001Test
https://figshare.com/articles/figure/KSHV_LANA_does_not_express_truncated_LANA_products_from_downstream_initiation_of_translation_/25015285Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: 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.

العلاقة: https://figshare.com/articles/dataset/Data_supporting_Figures_2_7_/25015294Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.s004Test
https://figshare.com/articles/dataset/Data_supporting_Figures_2_7_/25015294Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: Data supporting heatmaps in Figures 2 , 3 , 4 , 5 and 7 .

العلاقة: https://figshare.com/articles/dataset/Data_supporting_heatmaps_in_Figures_2_3_4_5_and_7_/25015297Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.s005Test
https://figshare.com/articles/dataset/Data_supporting_heatmaps_in_Figures_2_3_4_5_and_7_/25015297Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: A-H Ratios of viral RNA to DNA following KSHV or KSHVΔLANA infection of 293 cells for the indicated viral genes at 24, 48 or 72 hpi. ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001 by unpaired t test. I. Immunoblot of the indicated gene products following KSHV or KSHVΔLANA infection of 293 cells at 24 or 48 hpi at indicated MOI’s. The multiple LANA bands, which are more prominent at 48 hpi, are due to alternative initiation of translation and an alternative poly A signal.[ 71 , 72 ] GFP is expressed from the EF1α promoter in the recombinant viral genome. J. Gardella gel is shown at left. Phase microscopy (right, top) or detection of GFP expression by fluorescence microscopy (right, bottom) following infection of KSHV or KSHVΔLANA at 24 hpi. Cells were detected using the 4x objective on an EVOS M7000 imaging system (ThermoFisher). K. Heatmap of z score values showing relative viral gene expression for all KSHV genes following KSHV (WT) or KSHVΔLANA (ΔL) at 24 or 72 hpi in 293 cells. Three independent biological replicates are shown for each time point. Early lytic, late lytic, or latent classes of genes are indicated at left.

العلاقة: https://figshare.com/articles/figure/LANA_promotes_KSHV_gene_expression_in_293_cells_/25015309Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.g002Test
https://figshare.com/articles/figure/LANA_promotes_KSHV_gene_expression_in_293_cells_/25015309Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: 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.

العلاقة: https://figshare.com/articles/dataset/z_scores_used_for_heatmap_generation_/25015303Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.s007Test
https://figshare.com/articles/dataset/z_scores_used_for_heatmap_generation_/25015303Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: A. Immunoblot of LANA in 293FRT/LANA or BCBL1 PEL cells. 5x10 4 cells were loaded per lane. (BCBL1 PEL cells have less cytoplasm than do 293FRT cells, accounting for less tubulin in the BCBL1 lane.). B-I. Ratios of viral RNA to DNA following KSHVΔLANA infection of 293FRT/LANA or 293FRT cells for the indicated viral genes at 24, 48 or 72 hpi. **, p<0.01; ***, p<0.001; ****, p<0.0001 by unpaired t test. J. Heatmap of z score values showing relative viral gene expression for all KSHV genes following KSHVΔLANA infection of 293FRT or 293FRT LANA stably expressing cells at 24 or 72 hpi. Three independent biological replicates are shown for each time point. Early lytic, late lytic, or latent classes of genes are indicated at left. Gray color indicates absent values.

العلاقة: https://figshare.com/articles/figure/LANA_expressed_in_trans_promotes_KSHV_LANA_broad_viral_gene_transcription_following_infection_in_293FRT_cells_/25015312Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.g003Test
https://figshare.com/articles/figure/LANA_expressed_in_trans_promotes_KSHV_LANA_broad_viral_gene_transcription_following_infection_in_293FRT_cells_/25015312Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: A. Heatmap of z score values showing relative viral gene expression for all KSHV genes following KSHV (WT) or KSHVΔLANA (ΔL) at 24 or 72 hpi in SLK cells (derived from same data as in Fig 5J ), or iSLK cells that were induced for Rta expression. Three independent biological replicates are shown for each time point. Early lytic, late lytic, or latent classes of genes are indicated at left. Relative expression values were compared to the mean of all values for both SLK and iSLK for each gene, resulting in different z scores for SLK here compared to Fig 5J , which only included SLK. Gray color indicates absent values. B. Phase (top panels) or fluorescent microscopy (bottom panels) was performed to detect GFP expression from 293T cells 24 hours following incubation with supernatant from KSHV or KSHVΔLANA infected iSLK-RTA cells that had been induced for RTA expression. Cells were detected using the 4x objective on an EVOS M7000 imaging system (ThermoFisher). C. Viral titer of RTA induced, iSLK-RTA cell supernatant, 96 hpi following KSHV or KSHVΔLANA infection. GFP expression was detected by flow cytometry. D. Fold difference in KSHV genome copy number determined by qPCR of RTA induced, iSLK-RTA cell supernatant 96 hpi following KSHV or KSHVΔLANA infection.

العلاقة: https://figshare.com/articles/figure/RTA_expression_in_trans_leads_to_LANA_promotion_of_lytic_gene_expression_and_virus_production_in_SLK_cells_/25015324Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.g007Test
https://figshare.com/articles/figure/RTA_expression_in_trans_leads_to_LANA_promotion_of_lytic_gene_expression_and_virus_production_in_SLK_cells_/25015324Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: A. Gardella gel at 24 or 96 hpi at the indicated MOI following KSHV or KSHVΔLANA infection of SLK cells. Each lane pair contains independent infections. O, gel origin; E, episomal DNA; L, linear viral genomic DNA. B-I. Ratios of viral RNA to DNA following KSHV or KSHVΔLANA infection of SLK cells for the indicated viral genes at 24, 48 or 72 hpi. ns, p>0.05; *, p<0.05, **, p<0.01; ***, p<0.001;****, p<0.0001 by unpaired t test. J. Heatmap of z score values showing relative viral gene expression for all KSHV genes following KSHV (WT) or KSHVΔLANA (ΔL) at 24 or 72 hpi in SLK cells. Three independent biological replicates are shown for each time point. Early lytic, late lytic, or latent classes of genes are indicated at left.

العلاقة: https://figshare.com/articles/figure/KSHV_does_not_undergo_spontaneous_lytic_infection_following_infection_of_SLK_cells_and_LANA_inhibits_most_viral_gene_expression_/25015318Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.g005Test
https://figshare.com/articles/figure/KSHV_does_not_undergo_spontaneous_lytic_infection_following_infection_of_SLK_cells_and_LANA_inhibits_most_viral_gene_expression_/25015318Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: A. Gardella gel at 24 or 96 hpi at the indicated MOI following KSHV or KSHVΔLANA infection of NOK cells. Each lane pair contains independent infections. O, gel origin; E, episomal DNA; L, linear viral genomic DNA. B-I. Ratios of viral RNA to DNA following KSHV or KSHVΔLANA infection of NOK cells for the indicated viral genes at 24, 48 or 72 hpi. ns, p>0.05; *, p<0.05, **, p<0.01; ***, p<0.001;****, p<0.0001 by unpaired t test. J. Heatmap of z score values showing relative viral gene expression for all KSHV genes following KSHV (WT) or KSHVΔLANA (ΔL) at 24 or 72 hpi in NOK cells. Three independent biological replicates are shown for each time point. Early lytic, late lytic, or latent classes of genes are indicated at left.

العلاقة: https://figshare.com/articles/figure/KSHV_does_not_undergo_spontaneous_lytic_infection_following_infection_of_NOK_cells_and_LANA_inhibits_most_viral_gene_expression_/25015315Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.g004Test
https://figshare.com/articles/figure/KSHV_does_not_undergo_spontaneous_lytic_infection_following_infection_of_NOK_cells_and_LANA_inhibits_most_viral_gene_expression_/25015315Test

المؤلفون: Shijun Li, Mengbo Wang, Nicholas Van Sciver, Agnieszka Szymula, Vinayak Sadasivam Tumuluri, Athira George, Akshaya Ramachandran, Komal Raina, Catarina N. Costa, Bo Zhao, Majid Kazemian, J. Pedro Simas, Kenneth M. Kaye

مصطلحات موضوعية: Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Immunology, Cancer, Mental Health, Infectious Diseases, Plant Biology, Virology, virus gene specific, recombinant viral gfp, kaposi &# 8217, associated nuclear antigen, viral genome persistence, permissive cells led, cells partially permissive, lana &# 8217, lytic gene expression, latent gene expression, forced rta expression, sarcoma herpesvirus latency, lana promoted expression, permissive cells, sarcoma herpesvirus, repressed expression, lana promoted, antagonizing expression, genome maintenance, kshv latency

الوصف: 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.

العلاقة: https://figshare.com/articles/dataset/Oligonucleotides_used_for_PCR_amplification_/25015291Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1011907.s003Test
https://figshare.com/articles/dataset/Oligonucleotides_used_for_PCR_amplification_/25015291Test