Transcription of the herpes simplex virus 1 genome during productive and quiescent infection of neuronal and nonneuronal cells

Abstract

Herpes simplex virus 1 (HSV-1) can undergo a productive infection in nonneuronal and neuronal cells such that the genes of the virus are transcribed in an ordered cascade. HSV-1 can also establish a more quiescent or latent infection in peripheral neurons, where gene expression is substantially reduced relative to that in productive infection. HSV mutants defective in multiple immediate early (IE) gene functions are highly defective for later gene expression and model some aspects of latency in vivo. We compared the expression of wild-type (wt) virus and IE gene mutants in nonneuronal cells (MRC5) and adult murine trigeminal ganglion (TG) neurons using the Illumina platform for cDNA sequencing (RNA-seq). RNA-seq analysis of wild-type virus revealed that expression of the genome mostly followed the previously established kinetics, validating the method, while highlighting variations in gene expression within individual kinetic classes. The accumulation of immediate early transcripts differed between MRC5 cells and neurons, with a greater abundance in neurons. Analysis of a mutant defective in all five IE genes (d109) showed dysregulated genome-wide low-level transcription that was more highly attenuated in MRC5 cells than in TG neurons. Furthermore, a subset of genes in d109 was more abundantly expressed over time in neurons. While the majority of the viral genome became relatively quiescent, the latency-associated transcript was specifically upregulated. Unexpectedly, other genes within repeat regions of the genome, as well as the unique genes just adjacent the repeat regions, also remained relatively active in neurons. The relative permissiveness of TG neurons to viral gene expression near the joint region is likely significant during the establishment and reactivation of latency.

Importance: During productive infection, the genes of HSV-1 are transcribed in an ordered cascade. HSV can also establish a more quiescent or latent infection in peripheral neurons. HSV mutants defective in multiple immediate early (IE) genes establish a quiescent infection that models aspects of latency in vivo. We simultaneously quantified the expression of all the HSV genes in nonneuronal and neuronal cells by RNA-seq analysis. The results for productive infection shed further light on the nature of genes and promoters of different kinetic classes. In quiescent infection, there was greater transcription across the genome in neurons than in nonneuronal cells. In particular, the transcription of the latency-associated transcript (LAT), IE genes, and genes in the unique regions adjacent to the repeats persisted in neurons. The relative activity of this region of the genome in the absence of viral activators suggests a more dynamic state for quiescent genomes persisting in neurons.

FIG 1

Evaluation of RNA-seq reads from infected MRC5 cells. Monolayers of MRC5 cells and cultures of adult trigeminal neurons were infected with HSV-1 strain KOS at an MOI of 10 PFU/cell. Total RNA was collected from MRC5 cells at 1, 2, 3, 4, 6, 8, 12, and 16 h postinfection and from TG neurons at 2, 4, and 8 h postinfection. cDNA was prepared and subjected to Illumina sequencing as described in Materials and Methods. Illumina reads were processed and aligned to a modified KOS sequence using the TopHat mapper in the Galaxy Cloud software package. (A) Percentage of sequencing reads mapping to the HSV genome relative to the total number of reads. (B) Comparison of kinetics of ICP27 (UL54) accumulation by RNA-seq and RT-PCR. (C) Comparison of kinetics of tk (UL23) accumulation by RNA-seq and RT-PCR. (D) Comparison of kinetics of gC (UL44) accumulation by RNA-seq and RT-PCR. The units for RT-PCR were cDNA copies per μg RNA as determined by comparison to standards using CsCl-purified viral genomic DNA. The units for RNA-seq are reads mapped to the viral genome per million total reads (viral plus cell) per kilobase pair (MR per MTR per kb).

FIG 2

Locations of RNA-seq reads across the HSV genome as a function of time postinfection in MRC5 cells. The locations of reads for each of the time points is shown relative to a map of the mRNA for each of the HSV genes. The scales of the graphs were set to normalize to the number of total reads to enable qualitative comparison of transcript abundance between different time points. The reads and the genome are represented without the long and short terminal repeats since their sequences are represented within the internal repeats. The exons of RL2 and UL15 are also shown by darker shading.

FIG 3

Locations of RNA-seq reads across the HSV genome as a function of time postinfection in cultured TG neurons, as described in the legend for Fig. 2.

FIG 4

Accumulation of HSV transcripts in MRC5 cells and trigeminal neurons. Quantification of IE (α) (A and B), E (β) (C and D), L (γ1) (E and F), and L (γ2) (G and H) transcripts in MRC5 cells (A, C, E, and G) and neurons (B, D, F, and H) is shown. The units for RNA-seq are reads mapped to the viral genome per million total reads (viral plus cell) per kilobase pair (MR per MTR per kb).

FIG 5

Transcript accumulation in IE mutant-infected MRC5 cells. MRC5 cells were infected with KOS, n12, d106, and d109 at an MOI of 10 PFU/cell for 4 h and processed for RNA-seq as described in Materials and Methods. (A) Locations of mapped reads relative to the KOS genome. The Illumina reads were aligned to the modified KOS sequence as before. The maximum on the y axis is 30,000 reads. (B) Quantification of the numbers of reads for select HSV genes.

FIG 6

Percent viral reads in d109-infected MRC5 cells and neurons. The numbers of reads that mapped to the d109 genome were divided by the total number of reads and multiplied by 100.

FIG 7

HSV transcripts synthesized in d109-infected MRC5 cells and TG neurons. MRC cells and cultures of TG neurons were infected with d109 (MOI = 10 PFU/cell) for 4 h, 8 h, 24 h, and 7 days. The RNA synthesized in the cells was analyzed by RNA-seq as before, and the reads were mapped to a modified d109 genome. The reads were aligned to the d109 genome. The GFP gene is shown in red in the deleted ICP27 locus. The maximum on the y axes of the graphs was set to 100 reads. The gray shaded areas indicate regions where there were no reads.

FIG 8

Transcription of the internal repeat region from persisting genomes. (A) The RNA-seq reads from d109-infected TG neurons mapping to the region encoding the LAT promoter to US2 are shown relative to features in this region of the genome. The XbaI site at 111,151 marks the locus of the ICP0 deletion, and the site 113,549 marks the locus of the deletion that removed the VP16-responsive elements from the region. The numbers shown are in bp from the 5′ end of the modified d109 genome in the parental orientation. The maximum of the y axes for the 4- and 8-h graphs was set at 7,200 reads, while that for the 24-h and 7-day graphs was set at 1,800 reads. (B) The RNA-seq reads for the 1- and 7-day d109-infected TG neuron samples are shown in the region of the LAT TATA box, mRNA start sites, and ICP4 binding site. The maximum on the y axis is 100 reads. The beginning of the signal corresponds to the arrow to the left in the 7-day graph of panel A. (C) Quantification of the reads from the samples in Fig. 5 for select viral genes.