Functional Analysis of a Frontal miRNA Cluster Located in the Large Latency Transcript of Pseudorabies Virus

Abstract

MicroRNAs (miRNAs) have been identified as a class of crucial regulators of virus-host crosstalk, modulating such processes as viral replication, antiviral immune response, viral latency, and pathogenesis. Pseudorabies virus (PRV), a model for the study of alphaherpesvirus biology, codes for 11 distinct miRNAs mapped to the ~4.6 kb intron of Large Latency Transcript (LLT). Recent studies have revealed the role of clusters consisting of nine and eleven miRNA genes in the replication and virulence of PRV. The function of separate miRNA species in regulating PRV biology has not been thoroughly investigated. To analyze the regulatory potential of three PRV miRNAs located in the frontal cluster of the LLT intron, we generated a research model based on the constitutive expression of viral miRNAs in swine testis cells (ST_LLT [1-3] cell line). Using a cell culture system providing a stable production of individual miRNAs at high levels, we demonstrated that the LLT [1-3] miRNA cluster significantly downregulated IE180, EP0, and gE at the early stages of PRV infection. It was further determined that LLT [1-3] miRNAs could regulate the infection process, leading to a slight distortion in transmission and proliferation ability. Collectively, our findings indicate the potential of LLT [1-3] miRNAs to retard the host responses by reducing viral antigenic load and suppressing the expansion of progeny viruses at the early stages of infection.

Keywords: EP0; IE180; Large Latency Transcript; glycoprotein E; miRNA cluster; pseudorabies virus.

Figure 1

Characteristics of the ST_LLT [1–3] cell line constitutively expressing PRV miRNAs. (A) Schematic map of PRV-NIA-3 genome containing unique (UL and US) and inverted repeat (IR and TR) sequences. The enlarged section shows the border region between UL and IR, with loci for the EP0 and IE180 genes. The loci for prv-miR-LLTs (numbered from 1 to 11) are indicated by arrows. (B) Schematic map of the prv-miR-LLT [1–3] expression cassette. The LLT [1–3] miRNA cluster was introduced to the pLZRS-IRES-GFP retroviral vector, containing terminal repeats (5′LTR and 3′LTR), a packaging signal (Ψ), an internal ribosome entry site (IRES), followed by eGFP-encoding sequence. (C) Overexpression of PRV miRNAs in ST_LLT [1–3] cell line was confirmed by RT-qPCR. Fold differences of prv-miR-LLT1-3p, prv-miR-LLT2-5p, and prv-miR-LLT3-3p levels, relative to the U6 internal control, were determined with RT-qPCR using the ∆∆C_t method (with normalization of the relative miRNA expression to that of control ST cells). miRNA expression is depicted as mean values from three independent quantifications. (D) The PRV miRNAs effect on cell viability and proliferation was measured by the cell proliferation assay. The ST_LLT [1–3] or control ST cells were analyzed in 8-fold repeats (means with standard deviations are provided). Both cell lines were also treated with 10% dimethyl sulfoxide (DMSO) as a control.

Figure 2

Production of PRV LLT [1–3] miRNAs during PRV infection in cultured cells. The ST cells were infected with PRV at the multiplicity of infection (MOI) of 1 and collected at 3, 5, 7, and 24 hpi. The expression profiles of (A) LLT1, (B) LLT2, and (C) LLT3 in ST cells were assessed by RT-qPCR against mock-infected ST cells (time point 0). (D) Pattern of PRV LLT [1–3] miRNAs appearance in the course of infection. The data were reported as mean ± SD; the experiments were performed in triplicates.

Figure 3

PRV LLT [1–3] miRNAs downregulate the expression of IE180 and EP0. (A) The ST_LLT [1–3] cells and control ST cells were infected with PRV and IE180 protein level was determined by immunoblotting: at 3 hpi (160 µg of total protein extract per well), 5 hpi, and 7 hpi (40 µg of total protein extract per well). β-actin was detected as a loading control. (B) Densitometric quantifications of IE180 bands (indicated as the density ratio), normalized to the density of corresponding β-actin bands, were calculated between the control and ST_LLT [1–3] cell lines for each time point after PRV infection. n/a, not applicable. The mRNA levels of B. IE180 and (C) EP0 were measured by RT-qPCR at the indicated time points in the ST_LLT [1–3] cells and the control ST cells after PRV infection. 28S gene was used as a reference internal control. The experiment was performed in triplicate, data are expressed as the mean ± standard deviation. ** p < 0.01, *** p < 0.001.

Figure 4

PRV-infected ST_LLT [1–3] cells exhibit decreased gE expression (A) The RT-qPCR analysis of US8 transcript in the ST_LLT [1–3] cells infected with PRV, compared to the control ST cells. 28S gene was used as a reference internal control. mRNA expression is depicted as mean values from three independent quantifications. *** p < 0.001. (B) The immunoblot analysis at 5, and 7 hpi of PRV gE using lysates of the ST_LLT [1–3] cells and the control ST cells infected with PRV. β-actin was detected as a loading control. Densitometric quantifications of gE bands (indicated as the density ratio), normalized to the density of corresponding β-actin bands, were calculated between the control and ST_LLT [1–3] cell lines for each time point after PRV infection; n/a: not applicable. (C) The effect of LLT [1–3] miRNAs on the surface expression of gE in ST_LLT [1–3] cells was assessed by flow cytometry at 5, and 7 hpi and compared to uninfected control ST cells. The analysis was performed in triplicates. ** p < 0.01. (D) The level of subcellular gE in PRV-infected cells was evaluated by immunofluorescence laser scanning confocal microscopy (with the same image collection and analysis settings for all the samples).

Figure 5

Overexpression of PRV LLT [1–3] miRNAs affects PRV replication kinetics (A). The ST_LLT [1–3] cells and the control ST cells were infected with PRV and the plaque sizes of replicating PRV was analyzed. One-step growth kinetics of intracellular (B) and extracellular (C) virus replicating in the PRV-infected ST_LLT [1–3] cells compared to the control ST cells. The viral titers were determined on the ST cells using the plaque assay; * p < 0.05.