Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation

Abstract

The key events in herpes simplex virus (HSV) infections are (i) replication at a portal of entry into the body modeled by infection of cultured cells; (ii) establishment of a latent state characterized by a sole latency-associated transcript and microRNAs (miRNAs) modeled in murine peripheral ganglia 30 d after inoculation; and (iii) reactivation from the latent state modeled by excision and incubation of ganglia in medium containing anti-NGF antibody for a timespan of a single viral replicative cycle. In this report, we examine the pattern of synthesis and accumulation of 18 HSV-1 miRNAs in the three models. We report the following: (i) H2-3P, H3-3P, H4-3P, H5-3P, H6-3P, and H7-5P accumulated in ganglia harboring latent virus. All but H4-3P were readily detected in productively infected cells, and most likely they originate from three transcriptional units. (ii) H8-5P, H15, H17, H18, H26, and H27 accumulated during reactivation. Of this group, only H26 and H27 could be detected in productively infected cells. (iii) Of the 18 we have examined, only 10 miRNAs were found to accumulate above background levels in productively infected cells. The disparity in the accumulation of miRNAs in cell culture and during reactivation may reflect differences in the patterns of regulation of viral gene expression during productive infection and during reactivation from the latent state.

Keywords: alpha genes; beta/gamma genes; regulation of gene expression; transcription.

Fig. 1.

Detectable HSV-1 miRNAs in infected cells. Replicate cultures of Vero cells were exposed to 10 pfu of HSV-1(F) per cell and were mock-treated or incubated in medium containing cycloheximide (100 μg/mL) collected at 0, 1, 3, 6, and 12 h after infection. A replicate set of cultures was exposed to actinomycin D (10 μg/mL) at the time and was harvested at 0, 3 and 6 h after infection. HSV-1 miRNAs normalized with respect to the cellular miRNA Let-7a are shown as fold change compared with miRNAs detected in uninfected cells (0 h). They are shown as a function of time after infection. Change folds were plotted against hours after infection. (A and B) Group 1: pre-α miRNAs. (C and D) Group 2: α miRNAs. (E–J) Group 3: β/γ miRNAs.

Fig. 2.

(A–H) Group 4: undetectable miRNAs in infected cells. The experimental design is identical to that shown in Fig. 1.

Fig. 3.

HSV-1 miRNA expression in mice TG in latency and reactivation. At 30 d after inoculation, mice TG were excised and either analyzed immediately or incubated in medium containing anti-NGF antibody for 24 h. The procedures for extraction and assays were as described in Materials and Methods. HSV-1 miRNAs normalized by cellular miRNA Let-7a were compared with miRNAs detected in uninfected mice, presented as geometric means ± SD based on six samples per group. Student t test was performed to compare groups. The P values calculated by Student t test are shown. Two-tailed P value of 0.05 or less was considered statistically significant. (A) Detected in ganglia harboring latent virus. (B) Detected in ganglia 24 h after excision of ganglia. (C) Not detected in significant amounts.

Fig. 4.

Pattern of organization and expression of HSV-1 miRNAs. Shown are the locations of coding domains of relevant genes, miRNAs reported in these studies, and key cis-acting sites (A–D). The functions encoded in DNA strand from left to right are shown in blue above the dashed line, and the functions encoded in the strand from right to left are shown in red below the dashed line. The position of the sequences encoding the specific functions or cis-sites (e.g., Ori_S, Ori_L, or the a sequence) are for the HSV-1(F) strain and may be different for other HSV strains. The locations of miRNA precursors in the viral genomes are shown as arrowheads. The notations L, R, U, and n/a inside the arrowheads refer to detection of these miRNAs in mice ganglia: L, present in ganglia harboring latent virus; R, present in the ganglia incubated in medium containing anti-NGF antibody; U, undetected in ganglia; n/a, not available. Letters under arrowheads refer to miRNAs detected from lytic infected cells: pα (pre-α), α, and β/γ indicate the kinetic class of expression of viral genes; U, undetected. (C and D) For contextual completeness, the location of H14 is shown. H14 is antisense to H2. Its presence has not been verified in this study.