Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia - PubMed (original) (raw)

Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia

Jennifer L Umbach et al. J Virol. 2009 Oct.

Abstract

Analysis of cells infected by a wide range of herpesviruses has identified numerous virally encoded microRNAs (miRNAs), and several reports suggest that these viral miRNAs are likely to play key roles in several aspects of the herpesvirus life cycle. Here we report the first analysis of human ganglia for the presence of virally encoded miRNAs. Deep sequencing of human trigeminal ganglia latently infected with two pathogenic alphaherpesviruses, herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), confirmed the expression of five HSV-1 miRNAs, miR-H2 through miR-H6, which had previously been observed in mice latently infected with HSV-1. In addition, two novel HSV-1 miRNAs, termed miR-H7 and miR-H8, were also identified. Like four of the previously reported HSV-1 miRNAs, miR-H7 and miR-H8 are encoded within the second exon of the HSV-1 latency-associated transcript. Although VZV genomic DNA was readily detectable in the three human trigeminal ganglia analyzed, we failed to detect any VZV miRNAs, suggesting that VZV, unlike other herpesviruses examined so far, may not express viral miRNAs in latently infected cells.

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Figures

FIG. 1.

FIG. 1.

Two novel HSV-1 miRNAs recovered from latently infected humans. (A) Predicted secondary structures of the primary miRNA stem-loops of the novel HSV-1 miRNAs miR-H7 and miR-H8. Arrowheads indicate sites of Drosha cleavage, and arrows indicate sites of Dicer cleavage. The recovered, mature 5p and 3p miRNA strands are shown in boldface. (B) Schematic of the HSV-1 genome expanded to show details of the LAT locus. Relative sizes, locations, and orientations of other viral transcripts are also indicated. Sequence coordinates of viral miRNAs are given according to the HSV-1 strain 17 syn+ genome (NC_001806). IR, internal repeat; TR, terminal repeat; UL, unique long; US, unique short. The novel HSV-1 miRNAs miR-H7 and miR-H8 both map antisense to an intronic region in the viral ICP0 gene.

FIG. 2.

FIG. 2.

HSV-1 miRNA expression determined by qRT-PCR in latently and productively infected cells and tissues. All values are given as changes in expression relative to that of a non-HSV-1-infected control sample and were normalized to values for a cellular miRNA, miR-16. (A) HSV-1 miRNA expression levels in latently infected human TG from subject 1 (filled bars) and subject 2 (shaded bars). Despite its recovery during deep sequencing, miR-H8 was not detected in either TG sample. Subject 3 served as the negative control. (B) HSV-1 miRNA expression profiles, at 6 hpi (solid bars) and 18 hpi (shaded bars), of SY5Y cells productively infected with HSV-1 strain KOS at a multiplicity of infection of 10. (C and D) Similar to panel B, except that results for HeLa cells at 18 hpi are shown. (D) Similar to panel B, except that Vero cells at 24 hpi were analyzed.

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