Stem-loop RT-qPCR for miRNAs.

This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. miRNAs are 17 to 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least two times the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (T(m)) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe T(m) is optimized by addition of a minor groove binding (MGB) moiety.

Herpes simplex virus 1 microRNAs expressed abundantly during latent infection are not essential for latency in mouse trigeminal ganglia.

Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it.

Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2.

Certain viruses use microRNAs (miRNAs) to regulate the expression of their own genes, host genes, or both. Previous studies have identified a limited number of miRNAs expressed by herpes simplex viruses 1 and 2 (HSV-1 and -2), some of which are conserved between these two viruses. To more comprehensively analyze the miRNAs expressed by HSV-1 or HSV-2 during productive and latent infection, we applied a massively parallel sequencing approach. We were able to identify 16 and 17 miRNAs expressed by HSV-1 and HSV-2, respectively, including all previously known species, and a number of previously unidentified virus-encoded miRNAs. The genomic positions of most miRNAs encoded by these two viruses are within or proximal to the latency-associated transcript region. Nine miRNAs are conserved in position and/or sequence, particularly in the seed region, between these two viruses. Interestingly, we did not detect an HSV-2 miRNA homolog of HSV-1 miR-H1, which is highly expressed during productive infection, but we did detect abundant expression of miR-H6, whose seed region is conserved with HSV-1 miR-H1 and might represent a functional analog. We also identified a highly conserved miRNA family arising from the viral origins of replication. In addition, we detected several pairs of complementary miRNAs and we found miRNA-offset RNAs (moRs) arising from the precursors of HSV-1 and HSV-2 miR-H6 and HSV-2 miR-H4. Our results reveal elements of miRNA conservation and divergence that should aid in identifying miRNA functions.

MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs.

Herpesviruses are characterized by their ability to maintain life-long latent infections in their animal hosts. However, the mechanisms that allow establishment and maintenance of the latent state remain poorly understood. Herpes simplex virus 1 (HSV-1) establishes latency in neurons of sensory ganglia, where the only abundant viral gene product is a non-coding RNA, the latency associated transcript (LAT). Here we show that LAT functions as a primary microRNA (miRNA) precursor that encodes four distinct miRNAs in HSV-1 infected cells. One of these miRNAs, miR-H2-3p, is transcribed in an antisense orientation to ICP0-a viral immediate-early transcriptional activator that is important for productive HSV-1 replication and thought to have a role in reactivation from latency. We show that miR-H2-3p is able to reduce ICP0 protein expression, but does not significantly affect ICP0 messenger RNA levels. We also identified a fifth HSV-1 miRNA in latently infected trigeminal ganglia, miR-H6, which derives from a previously unknown transcript distinct from LAT. miR-H6 shows extended seed complementarity to the mRNA encoding a second HSV-1 transcription factor, ICP4, and inhibits expression of ICP4, which is required for expression of most HSV-1 genes during productive infection. These results may explain the reported ability of LAT to promote latency. Thus, HSV-1 expresses at least two primary miRNA precursors in latently infected neurons that may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression.

Prediction and identification of herpes simplex virus 1-encoded microRNAs.

MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. Recently, miRNAs have been identified from viruses with double-stranded DNA genomes. To attempt to identify miRNAs encoded by herpes simplex virus 1 (HSV-1), we applied a computational method to screen the complete genome of HSV-1 for sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. Using this method, we identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate, which is encoded approximately 450 bp upstream of the transcription start site of the latency-associated transcript (LAT), were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. Additionally, small RNAs, including some that are roughly the expected size of precursor miRNAs, were detected using probes for miRNA candidates derived from sequences encoding the 8.3-kilobase LAT, from sequences complementary to U(L)15 mRNA, and from the region between ICP4 and U(S)1. However, no species the size of typical mature miRNAs were detected using these probes. Three of these latter miRNA candidates were predicted to be conserved in HSV-2. Thus, HSV-1 encodes at least one miRNA. We hypothesize that HSV-1 miRNAs regulate viral and host gene expression.

High-throughput real-time quantitative reverse transcription PCR.

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Enzymatic amplification of DNA by PCR: standard procedures and optimization.

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection.

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1alpha, MIP-1beta and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.

Latent herpes simplex virus infection of sensory neurons alters neuronal gene expression.

The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.

The polymerase chain reaction.

This appendix describes the methodology behind PCR and gives a method for amplifying DNA enzymatically by the PCR and for optimizing this reaction for the sequence and primer set of interest.

Enzymatic amplification of DNA by PCR: standard procedures and optimization.

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl(2) concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer-template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl(2) concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation ("hot start" method), and reversible antibody binding are compared.