Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection Igor Jurak,a* Michael Hackenberg,b Ju Youn Kim,c Jean M. Pesola,a Roger D. Everett,d Chris M. Preston,d Angus C. Wilson,c Donald M. Coena Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USAa; Department of Genetics, University of Granada, Granada, Spainb; Department of Microbiology, New York University School of Medicine, New York, New York, USAc; MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotlandd
A
fter primary infection of mammalian hosts, herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in neurons of sensory ganglia (reviewed in reference 1). A hallmark of latency is the abundant expression of the latency-associated transcripts (LATs) and specific virus-encoded microRNAs (miRNAs; reviewed in references 1 and 2). Some HSV-1 miRNAs are expressed preferentially during productive (“lytic”) infection in cell culture and others during latent infection in sensory ganglia. For example, HSV-1 miR-H1 is abundantly expressed only during lytic infection, while miR-H2, -H3, and -H4 are most predominantly expressed during latency (3–6). An exception is HSV-1 miR-H6, which is expressed at high levels during both lytic and latent infection (3–5, 7, 8). There is evidence suggesting that certain HSV-1 miRNAs can repress the expression of important HSV-1 activators of lytic gene expression (7, 9). Addressing functions of miRNAs (and other gene products) for latent infection has been experimentally challenging, as studies of HSV latency have largely been limited to animal models. To address this challenge, cell culture models have been developed. To assess whether HSV-1 miRNA expression in three such models resembles that seen in latently infected ganglia, we extracted RNA, generated small-RNA libraries (TrueSeq small RNA sample preparation kit; Illumina), and sequenced the libraries on a HiSeq2000 sequencer (Illumina) at the Biopolymers Facility, Departments of Genetics, Harvard Medical School. Data sets were analyzed using miRanalyzer software (http://www.ncbi.nlm.nih.gov/pubmed/21515631) and aligned against known miRNAs from miRBase release 19 (10) by using Bowtie seed alignment, allowing for no mismatches within the first 18 nucleotides (11, 12). The total number of aligned sequence reads (read count) for each viral miRNA was normalized using read counts for cellular let-7a, whose expression does not change during HSV-1 infection (4, 6). Although the number of sequence reads does not necessarily reflect the actual abundance of a specific miRNA, it can be used to compare miRNA abundances among different samples (13–15). We analyzed a model employing primary human fetal foreskin fibroblasts (HFFF2; European Collection of Cell Cultures) infected at a multiplicity of infection (MOI) of ⬃3 at 38.5°C with in1374, a virus mutant derived from HSV-1 strain 17syn⫹ that carries mutations in genes encoding VP16, ICP0, and ICP4 (16– 18). Under these conditions, the virus exhibits negligible lytic gene
February 2014 Volume 88 Number 4
expression and does not produce infectious virus but can be reactivated by certain stimuli (18, 19). We analyzed infected cells at 1.5 and 16 h postinfection (hpi) (during establishment of quiescent infection) and at 8 days postinfection (dpi) (quiescent infection). We detected only four HSV-1 miRNAs (Fig. 1A). Of these, miR-H1 declined slightly during the experimental time frame, while miR-H4-5p increased slightly and miR-H3 and H4-3p increased substantially (Fig. 1A). Notably, we did not detect miR-H2 or miR-H6, which are relatively abundant in latently infected sensory ganglia (3, 4, 8) and whose expression is abolished by a 200-bp deletion that removes the LAT promoter (4). This expression pattern might be due in part to the activity of the long-short junction-spanning transcript promoter, which is ordinarily repressed by ICP4 (20, 21). In the second model, originally developed by McMahon and Walsh (22), serum-starved HFF cells were infected with wild-type (WT) HSV-1 strain KOS at an MOI of ⬃1 and incubated at 42°C until 72 hpi, at which point we detected no infectious virus or expression of the late protein, gC (data not shown). In this model, lytic replication of the virus can be triggered by exogenous expression of HSV-1 ICP0 (22). We detected expression of one or both (guide and passenger) strands of most HSV-1 miRNAs but at low levels (fewer than 10 reads), except for miR-H3 at 72 hpi and miR-H4-3p and miR-H6 at 18 and 72 hpi (Fig. 1B). There were more reads for miR-H1, a lytic miRNA, than for miR-H2, a latent miRNA. The expression of most miRNAs increased between 18 and 72 hpi, although miR-H6 levels increased only ⬃1.5-fold (Fig. 1B). Overall, this model appears to exhibit relatively low-level expression of both lytic and latent miRNAs. In the third model, we investigated primary neurons derived from rat superior cervical ganglia (SCG; obtained using an IACUC-
Received 26 November 2013 Accepted 26 November 2013 Published ahead of print 4 December 2013 Address correspondence to Donald M. Coen, [email protected]. * Present address: Igor Jurak, Department of Biotechnology, University of Rijeka, Rijeka, Croatia. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03486-13
Journal of Virology
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To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.
Jurak et al.
below the diagram, based on the number of detected sequence reads normalized to the number of reads representing let-7a in each group. (A) HFF2 cells quiescently infected with HSV-1 in1374. Black, gray, and white bars correspond to samples from 1 and 16 hpi and 8 dpi, respectively. (B) HFF cells quiescently infected with WT HSV-1 strain KOS at 42°C. Gray and white bars correspond to samples from 18 hpi and 72 hpi, respectively. (C) Rat SCG neurons latently infected with HSV-1 GFP-Us11 in the presence of NGF and ACV. White bars correspond to samples from 7 dpi.
approved protocol) that were infected with HSV-1 Patton strain GFP-Us11 at an MOI of 1 in the presence of acyclovir (ACV) and nerve growth factor (NGF) (23–25). Infectious virus and lytic gene expression are repressed, and virus can be reactivated by interruption of NGF signaling (23, 24). In latently infected neurons (7 dpi), we detected a relatively low number of reads for the lytic miRNA, miR-H1, and much higher numbers of reads for latent miRNAs H2, H3, H4, H6, H7, and H8 (Fig. 1C). The abundant expression of these miRNAs resembles the expression pattern previously found in HSV-1 latency in vivo (3–5, 7). In addition, the proportion of HSV-1 miRNAs in the total number of reads that could be aligned to the HSV-1 genome (mapped reads) was much higher in latently infected rat neurons (⬃0.2%) than in the other models (⬃0.01%) (data not shown). Moreover, in the rat neuron model, most HSV-1-specific reads corresponded to HSV-1 miRNAs, whereas in the other models we also detected a
large number of reads representing degradation products of lytic mRNAs (data not shown). Similarly, using quantitative stem-loop reverse transcription-PCR, as described previously (4), we found much higher levels of miR-H2 and miR-H4-3p than miR-H1 in this model, regardless of whether we used HSV-1 Patton strain GFP-Us11 or WT strains 17syn⫹ or KOS (Fig. 2). Taken together, these data indicate a program of miRNA expression in latently infected rat neurons in cell culture that appears more physiologically relevant to latency in vivo than that in the fibroblast systems. The more restricted program of gene expression in the in1374infected HFF2 model compared to that in the rat neuron model may reflect cell type differences in host factors important for expression of latency-specific miRNAs, greater repression of viral transcription generally in quiescently infected fibroblasts than in neurons (26), and/or, possibly, an alternative version of the latency program. Although cell culture models of latency cannot entirely reproduce in vivo conditions, such models are easily accessible, permit a relatively high MOI, and allow a variety of analyses that are extremely difficult in animal models. The complex nature of HSV-1 latency, including the expression of miRNAs, should be considered when selecting the appropriate model. The rat neuron model may be particularly useful for examining how mutations or oligonucleotides altering expression or function of latency-specific miRNAs affect parameters such as viral gene expression and reactivation. ACKNOWLEDGMENTS This work was supported in part by NIH grants P01 NS035138, R01 AI026126, R21 AI105896, and P01 AI098681 to D.M.C. and a collaborative project grant from the New York University Langone Medical Center (A.C.W.). J.Y.K. was supported by a Vilcek Endowment Fellowship Award and NIH grant T32AI07180. The work in the laboratories of R.D.E. and C.M.P. was supported by the Medical Research Council.
FIG 2 Similar expression of HSV-1 miRNAs in rat neurons latently infected with different virus strains. Rat SCG neurons were mock infected (mock) or latently infected with HSV-1 17syn⫹, KOS, or GFP-Us11 in the presence of NGF and ACV and assayed using quantitative reverse transcription-PCR for HSV-1 miR-H1 (H1; hatched bars), miR-H2 (H2; gray bars), and miR-H4-3p (H4-3p; black bars). Each bar represents the number of copies of the HSV-1 miRNAs normalized to the amount of host miRNA let-7a in the samples.
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REFERENCES 1. Roizman B, Knipe DM, Whitley RJ. 2013. Herpes simplex viruses, p 1823–1897. In Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (ed), Fields virology, 6th ed. Lippincott, Williams & Wilkins, Philadelphia, PA. 2. Jurak I, Griffiths A, Coen DM. 2011. Mammalian alphaherpesvirus
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FIG 1 HSV-1 miRNAs expressed in cell culture models of quiescent and latent infection. Each bar represents the relative expression of HSV-1 miRNA, indicated
HSV-1 miRNAs in Cell Culture Latency Models
3.
4.
5.
7.
8.
9.
10. 11.
12.
13.
14.
February 2014 Volume 88 Number 4
15.
16.
17. 18.
19. 20.
21.
22. 23.
24.
25.
26.
tory RNAs using cDNA library sequencing. Methods 44:3–12. http://dx .doi.org/10.1016/j.ymeth.2007.09.009. Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, Moller S, Litman T. 2009. Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing. RNA 15:2028 – 2034. http://dx.doi.org/10.1261/rna.1699809. Preston CM, Nicholl MJ. 2005. Human cytomegalovirus tegument protein pp71 directs long-term gene expression from quiescent herpes simplex virus genomes. J. Virol. 79:525–535. http://dx.doi.org/10.1128/JVI.79 .1.525-535.2005. Preston CM, Rinaldi A, Nicholl MJ. 1998. Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis. J. Gen. Virol. 79:117–124. Preston CM, Nicholl MJ. 2008. Induction of cellular stress overcomes the requirement of herpes simplex virus type 1 for immediate-early protein ICP0 and reactivates expression from quiescent viral genomes. J. Virol. 82:11775–11783. http://dx.doi.org/10.1128/JVI.01273-08. Preston CM. 2007. Reactivation of expression from quiescent herpes simplex virus type 1 genomes in the absence of immediate-early protein ICP0. J. Virol. 81:11781–11789. http://dx.doi.org/10.1128/JVI.01234-07. Lee LY, Schaffer PA. 1998. A virus with a mutation in the ICP4-binding site in the L/ST promoter of herpes simplex virus type 1, but not a virus with a mutation in open reading frame P, exhibits cell-type-specific expression of gamma(1)34.5 transcripts and latency-associated transcripts. J. Virol. 72:4250 – 4264. Yeh L, Schaffer PA. 1993. A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome. J. Virol. 67: 7373–7382. McMahon R, Walsh D. 2008. Efficient quiescent infection of normal human diploid fibroblasts with wild-type herpes simplex virus type 1. J. Virol. 82:10218 –10230. http://dx.doi.org/10.1128/JVI.00859-08. Camarena V, Kobayashi M, Kim JY, Roehm P, Perez R, Gardner J, Wilson AC, Mohr I, Chao MV. 2010. Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons. Cell Host Microbe 8:320 –330. http://dx.doi.org/10.1016/j .chom.2010.09.007. Kim JY, Mandarino A, Chao MV, Mohr I, Wilson AC. 2012. Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons. PLoS Pathog. 8:e1002540. http://dx.doi.org/10.1371/journal.ppat.1002540. Kobayashi M, Kim JY, Camarena V, Roehm PC, Chao MV, Wilson AC, Mohr I. 2012. A primary neuron culture system for the study of herpes simplex virus latency and reactivation. J. Vis. Exp. 62:e3823. http://dx.doi .org/10.3791/3823. Terry-Allison T, Smith CA, DeLuca NA. 2007. Relaxed repression of herpes simplex virus type 1 genomes in murine trigeminal ganglia. J. Virol. 81:12394 –12405. http://dx.doi.org/10.1128/JVI.01068-07.
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miRNAs. Biochim. Biophys. Acta 1809:641– 653. http://dx.doi.org/10 .1016/j.bbagrm.2011.06.010. Jurak I, Kramer MF, Mellor JC, van Lint AL, Roth FP, Knipe DM, Coen DM. 2010. Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2. J. Virol. 84:4659 – 4672. http://dx.doi.org /10.1128/JVI.02725-09. Kramer MF, Jurak I, Pesola JM, Boissel S, Knipe DM, Coen DM. 2011. Herpes simplex virus 1 microRNAs expressed abundantly during latent infection are not essential for latency in mouse trigeminal ganglia. Virology 417:239 –247. http://dx.doi.org/10.1016/j.virol.2011.06.027. Umbach JL, Nagel MA, Cohrs RJ, Gilden DH, Cullen BR. 2009. Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia. J. Virol. 83:10677–10683. http://dx.doi.org/10 .1128/JVI.01185-09. Cui C, Griffiths A, Li G, Silva LM, Kramer MF, Gaasterland T, Wang XJ, Coen DM. 2006. Prediction and identification of herpes simplex virus 1-encoded microRNAs. J. Virol. 80:5499 –5508. http://dx.doi.org/10.1128 /JVI.00200-06. Umbach JL, Kramer MF, Jura I, Karnowski HW, Coen DM, Cullen BR. 2008. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature 454:780 –783. http://dx.doi.org/10 .1038/nature07103. Du T, Zhou G, Roizman B. 2011. HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latencyassociated transcript and miRNAs. Proc. Natl. Acad. Sci. U. S. A. 108: 18820 –18824. http://dx.doi.org/10.1073/pnas.1117203108. Flores O, Nakayama S, Whisnant AW, Javanbakht H, Cullen BR, Bloom DC. 2013. Mutational inactivation of herpes simplex virus 1 microRNAs identifies viral mRNA targets and reveals phenotypic effects in culture. J. Virol. 87:6589 – 6603. http://dx.doi.org/10.1128/JVI.00504-13. Kozomara A, Griffiths-Jones S. 2011. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 39(Suppl 1): D152–D157. http://dx.doi.org/10.1093/nar/gkq1027. Hackenberg M, Sturm M, Langenberger D, Falcon-Perez JM, Aransay AM. 2009. miRanalyzer: a microRNA detection and analysis tool for nextgeneration sequencing experiments. Nucleic Acids Res. 37:W68 –W76. http://dx.doi.org/10.1093/nar/gkp347. Hackenberg M, Rodriguez-Ezpeleta N, Aransay AM. 2011. miRanalyzer: an update on the detection and analysis of microRNAs in highthroughput sequencing experiments. Nucleic Acids Res. 39:W132–W138. http://dx.doi.org/10.1093/nar/gkr247. Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J, Bertone P, Caldas C. 2010. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. RNA 16:991–1006. http: //dx.doi.org/10.1261/rna.1947110. Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, Holoch D, Lim C, Tuschl T. 2008. Identification of microRNAs and other small regula-
A
fter primary infection of mammalian hosts, herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in neurons of sensory ganglia (reviewed in reference 1). A hallmark of latency is the abundant expression of the latency-associated transcripts (LATs) and specific virus-encoded microRNAs (miRNAs; reviewed in references 1 and 2). Some HSV-1 miRNAs are expressed preferentially during productive (“lytic”) infection in cell culture and others during latent infection in sensory ganglia. For example, HSV-1 miR-H1 is abundantly expressed only during lytic infection, while miR-H2, -H3, and -H4 are most predominantly expressed during latency (3–6). An exception is HSV-1 miR-H6, which is expressed at high levels during both lytic and latent infection (3–5, 7, 8). There is evidence suggesting that certain HSV-1 miRNAs can repress the expression of important HSV-1 activators of lytic gene expression (7, 9). Addressing functions of miRNAs (and other gene products) for latent infection has been experimentally challenging, as studies of HSV latency have largely been limited to animal models. To address this challenge, cell culture models have been developed. To assess whether HSV-1 miRNA expression in three such models resembles that seen in latently infected ganglia, we extracted RNA, generated small-RNA libraries (TrueSeq small RNA sample preparation kit; Illumina), and sequenced the libraries on a HiSeq2000 sequencer (Illumina) at the Biopolymers Facility, Departments of Genetics, Harvard Medical School. Data sets were analyzed using miRanalyzer software (http://www.ncbi.nlm.nih.gov/pubmed/21515631) and aligned against known miRNAs from miRBase release 19 (10) by using Bowtie seed alignment, allowing for no mismatches within the first 18 nucleotides (11, 12). The total number of aligned sequence reads (read count) for each viral miRNA was normalized using read counts for cellular let-7a, whose expression does not change during HSV-1 infection (4, 6). Although the number of sequence reads does not necessarily reflect the actual abundance of a specific miRNA, it can be used to compare miRNA abundances among different samples (13–15). We analyzed a model employing primary human fetal foreskin fibroblasts (HFFF2; European Collection of Cell Cultures) infected at a multiplicity of infection (MOI) of ⬃3 at 38.5°C with in1374, a virus mutant derived from HSV-1 strain 17syn⫹ that carries mutations in genes encoding VP16, ICP0, and ICP4 (16– 18). Under these conditions, the virus exhibits negligible lytic gene
February 2014 Volume 88 Number 4
expression and does not produce infectious virus but can be reactivated by certain stimuli (18, 19). We analyzed infected cells at 1.5 and 16 h postinfection (hpi) (during establishment of quiescent infection) and at 8 days postinfection (dpi) (quiescent infection). We detected only four HSV-1 miRNAs (Fig. 1A). Of these, miR-H1 declined slightly during the experimental time frame, while miR-H4-5p increased slightly and miR-H3 and H4-3p increased substantially (Fig. 1A). Notably, we did not detect miR-H2 or miR-H6, which are relatively abundant in latently infected sensory ganglia (3, 4, 8) and whose expression is abolished by a 200-bp deletion that removes the LAT promoter (4). This expression pattern might be due in part to the activity of the long-short junction-spanning transcript promoter, which is ordinarily repressed by ICP4 (20, 21). In the second model, originally developed by McMahon and Walsh (22), serum-starved HFF cells were infected with wild-type (WT) HSV-1 strain KOS at an MOI of ⬃1 and incubated at 42°C until 72 hpi, at which point we detected no infectious virus or expression of the late protein, gC (data not shown). In this model, lytic replication of the virus can be triggered by exogenous expression of HSV-1 ICP0 (22). We detected expression of one or both (guide and passenger) strands of most HSV-1 miRNAs but at low levels (fewer than 10 reads), except for miR-H3 at 72 hpi and miR-H4-3p and miR-H6 at 18 and 72 hpi (Fig. 1B). There were more reads for miR-H1, a lytic miRNA, than for miR-H2, a latent miRNA. The expression of most miRNAs increased between 18 and 72 hpi, although miR-H6 levels increased only ⬃1.5-fold (Fig. 1B). Overall, this model appears to exhibit relatively low-level expression of both lytic and latent miRNAs. In the third model, we investigated primary neurons derived from rat superior cervical ganglia (SCG; obtained using an IACUC-
Received 26 November 2013 Accepted 26 November 2013 Published ahead of print 4 December 2013 Address correspondence to Donald M. Coen, [email protected]. * Present address: Igor Jurak, Department of Biotechnology, University of Rijeka, Rijeka, Croatia. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03486-13
Journal of Virology
p. 2337–2339
jvi.asm.org
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Downloaded from http://jvi.asm.org/ on February 24, 2015 by UCSF Library & CKM
To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.
Jurak et al.
below the diagram, based on the number of detected sequence reads normalized to the number of reads representing let-7a in each group. (A) HFF2 cells quiescently infected with HSV-1 in1374. Black, gray, and white bars correspond to samples from 1 and 16 hpi and 8 dpi, respectively. (B) HFF cells quiescently infected with WT HSV-1 strain KOS at 42°C. Gray and white bars correspond to samples from 18 hpi and 72 hpi, respectively. (C) Rat SCG neurons latently infected with HSV-1 GFP-Us11 in the presence of NGF and ACV. White bars correspond to samples from 7 dpi.
approved protocol) that were infected with HSV-1 Patton strain GFP-Us11 at an MOI of 1 in the presence of acyclovir (ACV) and nerve growth factor (NGF) (23–25). Infectious virus and lytic gene expression are repressed, and virus can be reactivated by interruption of NGF signaling (23, 24). In latently infected neurons (7 dpi), we detected a relatively low number of reads for the lytic miRNA, miR-H1, and much higher numbers of reads for latent miRNAs H2, H3, H4, H6, H7, and H8 (Fig. 1C). The abundant expression of these miRNAs resembles the expression pattern previously found in HSV-1 latency in vivo (3–5, 7). In addition, the proportion of HSV-1 miRNAs in the total number of reads that could be aligned to the HSV-1 genome (mapped reads) was much higher in latently infected rat neurons (⬃0.2%) than in the other models (⬃0.01%) (data not shown). Moreover, in the rat neuron model, most HSV-1-specific reads corresponded to HSV-1 miRNAs, whereas in the other models we also detected a
large number of reads representing degradation products of lytic mRNAs (data not shown). Similarly, using quantitative stem-loop reverse transcription-PCR, as described previously (4), we found much higher levels of miR-H2 and miR-H4-3p than miR-H1 in this model, regardless of whether we used HSV-1 Patton strain GFP-Us11 or WT strains 17syn⫹ or KOS (Fig. 2). Taken together, these data indicate a program of miRNA expression in latently infected rat neurons in cell culture that appears more physiologically relevant to latency in vivo than that in the fibroblast systems. The more restricted program of gene expression in the in1374infected HFF2 model compared to that in the rat neuron model may reflect cell type differences in host factors important for expression of latency-specific miRNAs, greater repression of viral transcription generally in quiescently infected fibroblasts than in neurons (26), and/or, possibly, an alternative version of the latency program. Although cell culture models of latency cannot entirely reproduce in vivo conditions, such models are easily accessible, permit a relatively high MOI, and allow a variety of analyses that are extremely difficult in animal models. The complex nature of HSV-1 latency, including the expression of miRNAs, should be considered when selecting the appropriate model. The rat neuron model may be particularly useful for examining how mutations or oligonucleotides altering expression or function of latency-specific miRNAs affect parameters such as viral gene expression and reactivation. ACKNOWLEDGMENTS This work was supported in part by NIH grants P01 NS035138, R01 AI026126, R21 AI105896, and P01 AI098681 to D.M.C. and a collaborative project grant from the New York University Langone Medical Center (A.C.W.). J.Y.K. was supported by a Vilcek Endowment Fellowship Award and NIH grant T32AI07180. The work in the laboratories of R.D.E. and C.M.P. was supported by the Medical Research Council.
FIG 2 Similar expression of HSV-1 miRNAs in rat neurons latently infected with different virus strains. Rat SCG neurons were mock infected (mock) or latently infected with HSV-1 17syn⫹, KOS, or GFP-Us11 in the presence of NGF and ACV and assayed using quantitative reverse transcription-PCR for HSV-1 miR-H1 (H1; hatched bars), miR-H2 (H2; gray bars), and miR-H4-3p (H4-3p; black bars). Each bar represents the number of copies of the HSV-1 miRNAs normalized to the amount of host miRNA let-7a in the samples.
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REFERENCES 1. Roizman B, Knipe DM, Whitley RJ. 2013. Herpes simplex viruses, p 1823–1897. In Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (ed), Fields virology, 6th ed. Lippincott, Williams & Wilkins, Philadelphia, PA. 2. Jurak I, Griffiths A, Coen DM. 2011. Mammalian alphaherpesvirus
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FIG 1 HSV-1 miRNAs expressed in cell culture models of quiescent and latent infection. Each bar represents the relative expression of HSV-1 miRNA, indicated
HSV-1 miRNAs in Cell Culture Latency Models
3.
4.
5.
7.
8.
9.
10. 11.
12.
13.
14.
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15.
16.
17. 18.
19. 20.
21.
22. 23.
24.
25.
26.
tory RNAs using cDNA library sequencing. Methods 44:3–12. http://dx .doi.org/10.1016/j.ymeth.2007.09.009. Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, Moller S, Litman T. 2009. Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing. RNA 15:2028 – 2034. http://dx.doi.org/10.1261/rna.1699809. Preston CM, Nicholl MJ. 2005. Human cytomegalovirus tegument protein pp71 directs long-term gene expression from quiescent herpes simplex virus genomes. J. Virol. 79:525–535. http://dx.doi.org/10.1128/JVI.79 .1.525-535.2005. Preston CM, Rinaldi A, Nicholl MJ. 1998. Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis. J. Gen. Virol. 79:117–124. Preston CM, Nicholl MJ. 2008. Induction of cellular stress overcomes the requirement of herpes simplex virus type 1 for immediate-early protein ICP0 and reactivates expression from quiescent viral genomes. J. Virol. 82:11775–11783. http://dx.doi.org/10.1128/JVI.01273-08. Preston CM. 2007. Reactivation of expression from quiescent herpes simplex virus type 1 genomes in the absence of immediate-early protein ICP0. J. Virol. 81:11781–11789. http://dx.doi.org/10.1128/JVI.01234-07. Lee LY, Schaffer PA. 1998. A virus with a mutation in the ICP4-binding site in the L/ST promoter of herpes simplex virus type 1, but not a virus with a mutation in open reading frame P, exhibits cell-type-specific expression of gamma(1)34.5 transcripts and latency-associated transcripts. J. Virol. 72:4250 – 4264. Yeh L, Schaffer PA. 1993. A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome. J. Virol. 67: 7373–7382. McMahon R, Walsh D. 2008. Efficient quiescent infection of normal human diploid fibroblasts with wild-type herpes simplex virus type 1. J. Virol. 82:10218 –10230. http://dx.doi.org/10.1128/JVI.00859-08. Camarena V, Kobayashi M, Kim JY, Roehm P, Perez R, Gardner J, Wilson AC, Mohr I, Chao MV. 2010. Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons. Cell Host Microbe 8:320 –330. http://dx.doi.org/10.1016/j .chom.2010.09.007. Kim JY, Mandarino A, Chao MV, Mohr I, Wilson AC. 2012. Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons. PLoS Pathog. 8:e1002540. http://dx.doi.org/10.1371/journal.ppat.1002540. Kobayashi M, Kim JY, Camarena V, Roehm PC, Chao MV, Wilson AC, Mohr I. 2012. A primary neuron culture system for the study of herpes simplex virus latency and reactivation. J. Vis. Exp. 62:e3823. http://dx.doi .org/10.3791/3823. Terry-Allison T, Smith CA, DeLuca NA. 2007. Relaxed repression of herpes simplex virus type 1 genomes in murine trigeminal ganglia. J. Virol. 81:12394 –12405. http://dx.doi.org/10.1128/JVI.01068-07.
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