Expression Profile of Human Cytomegalovirus UL111A cmvIL-10 and LAcmvIL-10 Transcripts in Primary Cells and Cells from Renal Transplant Recipients

Abstract

Human cytomegalovirus (HCMV) is a high-risk pathogen in immunocompromised individuals, especially in transplant recipients. HCMV viremia must be monitored, and frequently, patients are treated with antiviral agents. HCMV has a variety of strategies to modulate host antiviral responses, and one important player is a viral homolog of the cellular interleukin-10 (cIL-10). The viral UL111A gene produces several HCMV IL-10 transcripts and protein isoforms through alternative splicing. The cmvIL-10 (isoform A) has similar properties to cIL-10, while LAcmvIL-10 (isoform B) has more restricted biological properties. Other isoforms are produced (C to H) but have unknown functions. Here, we investigated the expression of the most abundant transcripts, cmvIL-10 and LAcmvIL-10, in productively and latently infected cells and in peripheral blood mononuclear cells from renal transplant recipients up to 60 days post-transplantation. This study investigated HCMV cmvIL-10 and LAcmvIL-10 transcription profiles in vitro, in productive and latent infection, and in vivo, in peripheral blood mononuclear cells (PBMCs) of renal transplant patients. In vitro, both cmvIL-10 and LAcmvIL-10 transcripts were detected in both types at high levels and low levels in MRC-5 and latent infected (CD14+). When PBMCs from transplant patients were analyzed, LAcmvIL-10 was detected mostly sporadically and in only a few patients, while cmvIL-10 was found in all patients at all time points. Furthermore, it was observed in PBMCs that expression of cmvIL-10 was positively associated with an increase in viral DNA detection in the subsequently collected sample, indicating that expression of cmvIL-10 might precede viral DNA replication. These results contribute to the understanding of HCMV biology in different phases of infection. In addition, our initial analysis suggests that monitoring cmvIL-10, along with viral DNA, could improve early detection of HCMV reactivation in transplant recipients.

Keywords: HCMV IL-10; HCMV latent infection; HCMV lytic infection; UL111A transcripts; human cytomegalovirus (HCMV); kidney transplant.

Figure 1

Analysis of UL111A cmvIL-10 and LAcmvIL-10 transcripts in permissive infected MRC-5 cells and during the establishment of latency in CD14+ monocytes. MRC-5 (A) and CD14+ monocytes (B) were infected with HCMV TB40E strain at MOI 1 and 5, respectively; cmvIL10 and LAcmvIL-10 RNA levels were quantified by RT-qPCR and are expressed as fold change relative to housekeeping gene GAPDH (on the Y-axis) at different time points: 24, 48, 72, and 96 hpi (on the X-axis). For viral reactivation in CD14+ monocytes, phorbolmyristate acetate (PMA) was added to the media at 96 hpi and left until 120 hpi. Each bar represents the means with standard deviations of fold changes in expression of the experimental results of three biological and two technical repeats for each time point (expression in CD14+ monocytes was investigated from 2 different donors, and bars represent the mean of both sets of data).

Figure 2

Relative expression of UL123 and UL138 genes in CD14+ cells. UL123 (immediate-early gene expressed only during lytic infection) and UL138 HCMV transcripts were quantified by RT-qPCR at 96 hpi during latency (A) and in the absence (latent) and in the presence of PMA treatment (reactivating) at 96 hpi (B) and at 120 hpi (C). Expression is shown as fold change relative to the housekeeping gene GAPDH of experimental results of three biological and two technical repeats (on the Y-axis).

Figure 3

Expression of cmvIL-10 and LAcmvIL-10 transcripts and intracellular viral DNA in PBMCs from transplant recipients. In each graph, the left Y- axis shows the expression of transcripts in copy numbers/µL; the right Y-axis shows the relative levels of intracellular DNA (UL44 gene) relative to GAPDH; and the X-axis shows the time points (days) of the analysis. The transcript expressions are shown in bars: transcript cmvIL10 (blue) and LAcmvIL-10 (red). Viral DNA is shown in black lines. The beginning and end of ganciclovir treatment are indicated as I.T. (initial treatment) and F.T. (final treatment).

Figure 4

Analysis of covariance between cmvIL-10 transcript expression and viral DNA. Covariance indicates the extent to which two (random) variables are dependent on each other. A positive covariance on a graph is represented as a line that slopes upwards. (A) Analysis of cmvIL-10 and viral DNA of samples from the same time point, i.e., analysis was made using data from the same sample; same time-point analysis did not show a significant covariance (Pr(>F) = 0.1976). (B) Analysis of cmvIL-10 and viral DNA in sequential samples, i.e., analysis of cmvIL-10 transcript expression was performed with one sample, and viral DNA expression was performed with a sample from a subsequent collection time point in that patient; subsequent collection analysis showed a statistically significant positive covariance (Pr(>F) = 0.0212). Circles represent the expression of cmvIL-10 transcript (in copies/μL, on the X-axis) and viral DNA levels (UL44 DNA levels relative to GAPDH, on the Y-axis) in the samples; lines represent the best straight line showing the relationship of cmvIL-10 concentration with viral DNA expression for each patient (each color represents a different patient). All samples were collected before ganciclovir treatment.