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Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Krishna BA, Lau B, Jackson SE, Wills MR, Sinclair JH, Poole E - Sci Rep (2016)

Bottom Line: Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle.We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation.In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Level 5 Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ.

ABSTRACT
Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

No MeSH data available.


Related in: MedlinePlus

MC1568 does not cause full reactivation and pan-HDAC inhibitors also induce IE expression.5 day latent (latent) monocytes were left untreated, treated with MC1568 for 7 days or differentiated. RNAs encoding viral IE, UL99 (pp28), UL138 and cellular GAPDH were analysed in duplicate by RT-qPCR and standard deviations are shown (A). Alternatively, 5 day latent monocytes (latent) were treated with MC1568 or differentiated and then co-cultured with fibroblasts to analyse infectious foci formation (B). 5 day latent monocytes were also treated over 1–7 days with VPA or TSA and analysed for IE expression. Data shown (6 replicates) are relative to the level of IE induced following reactivation by differentiation (C). Additionally, 5 day latent CD34+ cells (latent) were treated with MC1568, TSA or VPA for 24h (Day 1) and levels of IE expression were also analysed at days 2, 5 and 7 without removal of the drugs. Data are shown (6 replicates) relative to IE expression induced by differentiation of CD34+ cells. Mock represents IE expression in uninfected cells. Standard deviations are shown and statistical significance was determined using the Student’s t-test (D).
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f2: MC1568 does not cause full reactivation and pan-HDAC inhibitors also induce IE expression.5 day latent (latent) monocytes were left untreated, treated with MC1568 for 7 days or differentiated. RNAs encoding viral IE, UL99 (pp28), UL138 and cellular GAPDH were analysed in duplicate by RT-qPCR and standard deviations are shown (A). Alternatively, 5 day latent monocytes (latent) were treated with MC1568 or differentiated and then co-cultured with fibroblasts to analyse infectious foci formation (B). 5 day latent monocytes were also treated over 1–7 days with VPA or TSA and analysed for IE expression. Data shown (6 replicates) are relative to the level of IE induced following reactivation by differentiation (C). Additionally, 5 day latent CD34+ cells (latent) were treated with MC1568, TSA or VPA for 24h (Day 1) and levels of IE expression were also analysed at days 2, 5 and 7 without removal of the drugs. Data are shown (6 replicates) relative to IE expression induced by differentiation of CD34+ cells. Mock represents IE expression in uninfected cells. Standard deviations are shown and statistical significance was determined using the Student’s t-test (D).

Mentions: Interestingly, this induction of viral IE gene expression by MC1568 did not result in reactivation of the full lytic cycle, even after 7 days continuous treatment, as no expression of viral UL99 (pp28), a true late viral gene only expressed after viral DNA replication, was detected (Fig. 2A). Consistent with this, MC1568-treated monocytes showed no evidence of reactivation of infectious virus as detected by co-culture on indicator fibroblasts (Fig. 2B). Additionally, if latently infected monocytes were treated with other pan-specific HDAC inhibitors such as valproic acid (VPA) or trichostatin A (TSA), a transient induction of IE gene expression was also detectable (Fig. 2C). Interestingly, treatment of latently infected CD34+ progenitor cells with VPA and MC1568 also induced IE gene expression (Fig. 2D). However, consistent with reports that CD34+ cells are unresponsive to TSA18, induction of IE gene expression from latently infected CD34+ cells was not observed if cells were induced with TSA (Fig. 2D).


Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Krishna BA, Lau B, Jackson SE, Wills MR, Sinclair JH, Poole E - Sci Rep (2016)

MC1568 does not cause full reactivation and pan-HDAC inhibitors also induce IE expression.5 day latent (latent) monocytes were left untreated, treated with MC1568 for 7 days or differentiated. RNAs encoding viral IE, UL99 (pp28), UL138 and cellular GAPDH were analysed in duplicate by RT-qPCR and standard deviations are shown (A). Alternatively, 5 day latent monocytes (latent) were treated with MC1568 or differentiated and then co-cultured with fibroblasts to analyse infectious foci formation (B). 5 day latent monocytes were also treated over 1–7 days with VPA or TSA and analysed for IE expression. Data shown (6 replicates) are relative to the level of IE induced following reactivation by differentiation (C). Additionally, 5 day latent CD34+ cells (latent) were treated with MC1568, TSA or VPA for 24h (Day 1) and levels of IE expression were also analysed at days 2, 5 and 7 without removal of the drugs. Data are shown (6 replicates) relative to IE expression induced by differentiation of CD34+ cells. Mock represents IE expression in uninfected cells. Standard deviations are shown and statistical significance was determined using the Student’s t-test (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835774&req=5

f2: MC1568 does not cause full reactivation and pan-HDAC inhibitors also induce IE expression.5 day latent (latent) monocytes were left untreated, treated with MC1568 for 7 days or differentiated. RNAs encoding viral IE, UL99 (pp28), UL138 and cellular GAPDH were analysed in duplicate by RT-qPCR and standard deviations are shown (A). Alternatively, 5 day latent monocytes (latent) were treated with MC1568 or differentiated and then co-cultured with fibroblasts to analyse infectious foci formation (B). 5 day latent monocytes were also treated over 1–7 days with VPA or TSA and analysed for IE expression. Data shown (6 replicates) are relative to the level of IE induced following reactivation by differentiation (C). Additionally, 5 day latent CD34+ cells (latent) were treated with MC1568, TSA or VPA for 24h (Day 1) and levels of IE expression were also analysed at days 2, 5 and 7 without removal of the drugs. Data are shown (6 replicates) relative to IE expression induced by differentiation of CD34+ cells. Mock represents IE expression in uninfected cells. Standard deviations are shown and statistical significance was determined using the Student’s t-test (D).
Mentions: Interestingly, this induction of viral IE gene expression by MC1568 did not result in reactivation of the full lytic cycle, even after 7 days continuous treatment, as no expression of viral UL99 (pp28), a true late viral gene only expressed after viral DNA replication, was detected (Fig. 2A). Consistent with this, MC1568-treated monocytes showed no evidence of reactivation of infectious virus as detected by co-culture on indicator fibroblasts (Fig. 2B). Additionally, if latently infected monocytes were treated with other pan-specific HDAC inhibitors such as valproic acid (VPA) or trichostatin A (TSA), a transient induction of IE gene expression was also detectable (Fig. 2C). Interestingly, treatment of latently infected CD34+ progenitor cells with VPA and MC1568 also induced IE gene expression (Fig. 2D). However, consistent with reports that CD34+ cells are unresponsive to TSA18, induction of IE gene expression from latently infected CD34+ cells was not observed if cells were induced with TSA (Fig. 2D).

Bottom Line: Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle.We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation.In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Level 5 Laboratories Block, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ.

ABSTRACT
Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

No MeSH data available.


Related in: MedlinePlus