Human cytomegalovirus chemokine receptor gene US28 is transcribed in latently infected THP-1 monocytes

Abstract

The human cytomegalovirus (HCMV) US28 gene product, pUS28, is a G protein-coupled receptor that interacts with both CC and CX(3)C chemokines. To date, the role of pUS28 in immune evasion and cell migration has been studied only in cell types that can establish productive HCMV infection. We show that HCMV can latently infect THP-1 monocytes and that during latency US28 is transcribed. We also show that the transcription is sustained during differentiation of the THP-1 monocytes. Since cells expressing pUS28 were previously shown to adhere to immobilized CX(3)C chemokines (C. A. Haskell, M. D. Cleary, and I. F. Charo, J. Biol. Chem. 275:34183-34189, 2000), we hypothesize that latently infected circulating monocytes express pUS28, thereby enabling adhesion of these cells to CX(3)C-exposing endothelium. Consequently, the US28-encoded chemokine receptor may play an important role in dissemination of latent HCMV.

FIG. 1

RT-PCR primers and their target transcripts. Schematic representation of the HCMV Toledo genome and the relative positions of the MIE gene and US28. Black boxes represent repeat regions of the Toledo genome. The US28-specific transcripts and CLTs are indicated below the genome at a smaller scale by open arrows. The positions and polarity of the US28-, as well as the CLT-specific RT-PCR primers (Table 1) are indicated by black arrows. Transcription starts of the sense CLTs (LSS1 and LSS2) and the PSS are indicated by arrowheads.

FIG. 2

The US28 and MIE genes are transcribed in HCMV-infected THP-1 cells. The figure shows ethidium bromide-stained 2% agarose gels in which RT-PCR samples were separated. All agarose gel images shown in this report were digitized and contrast-inverted for clarity using a video scanner (Virbert Lourmat, Marne la Vallée, France). Molecular weight (MW) marker sizes are indicated on the left of each panel, and the primer sets corresponding to each panel are indicated on the right. Black arrowheads denote the relevant PCR products. (A) Detection of GAPDH-specific transcripts, antisense CLT-specific, and US28-specific transcripts in a panel of HCMV-infected myeloid cell types at day 8 p.i. The cell types used are indicated above the panel using the following abbreviations: H, HL-60; K5, K562; KG, KG1a; T, THP-1; U, U937. −RT, sample not treated with reverse transcriptase. (B) Detection of sense CLTs from HCMV-infected THP-1 cells at day 8 p.i. The primer combinations that were used in this experiment (see also Table 1 and Fig. 1) are indicated above each lane. (C) Detection of GAPDH-specific transcripts, anti-sense CLT-specific, and US28-specific transcripts in HCMV-infected THP-1 cells at day 15 p.i. M, mock infected; I, HCMV Toledo infected.

FIG. 3

US28-specific transcripts are deposited either inside or at the surface of THP-1 cells immediately after infection but transcribed de novo at later times p.i. RT-PCR samples are visualized on agarose gels as described in Fig. 2. (A) Detection of viral and cellular poly(A)⁺ RNA in virus inoculum from HCMV-infected fibroblasts. The primer sets that were included in the RT-PCR samples are indicated on top of the panel. (B) Detection of US28-specific transcripts either in actinomycin-D-treated HCMV-infected THP-1 cells or in THP-1 cells infected with UV-inactivated HCMV. The primer sets that were included in the RT-PCR samples are indicated at the right of each panel. Samples from which reverse transcriptase enzyme was omitted remained PCR negative (data not shown). Abbreviations: −RT, sample not treated with reverse transcriptase; +RT, reverse transcriptase treated; Δ, HCMV RV101 (US27-US28 deletion mutant) virions; WT, wild-type HCMV; h, hours p.i.; d, days p.i.; A, actinomycin D treated; UV, UV-treated; NT, not treated.

FIG. 4

Expression of lytic-phase antigens in HCMV-infected THP-1 cells at day 7 p.i. The top panels show immunocytometric histograms of HCMV-infected MRC5 fibroblasts (total cell count = 10,000) at day 2 p.i. The lower panels show histograms of infected THP-1 cells (total cell count = 500,000) at day 7 p.i. FL1-H, relative intensity of fluorescence. Dotted lines represent uninfected cells.

FIG. 5

Quantitative comparison of US28 and HCMV DNA polymerase gene transcription in HCMV-infected THP-1 cells at day 7 p.i. RT-PCR samples are visualized on agarose gels as described in Fig. 2. The primer sets that correspond to each of the panels are indicated on the right of each panel. Black arrowheads denote the relevant PCR products. (A) Sensitivity of RT-PCR for the detection of US28 and HCMV DNA polymerase cDNA. The amount of target cDNA molecules that was included in each RT-PCR sample is indicated at the top. (B) Quantification of HCMV-infected THP-1 cells transcribing either US28 or the HCMV DNA polymerase gene at day 7 p.i. The quantities on top correspond to the amount of cells that were taken from the original HCMV-infected THP-1 culture and mixed with uninfected THP-1 cells to a total of 2 × 10⁵ prior to cDNA preparation and RT-PCR. (C) Quantification of US28- and HCMV DNA polymerase-specific transcripts in untreated and PAA-treated HCMV-infected THP-1 cells at day 7 p.i. The dilution factor of each cDNA sample relative to an initial sample representing 2 × 10⁵ cells is indicated on the top. Abbreviations: M, mock-infected; I, HCMV-infected; I/PAA, infected and PAA-treated; −RT, sample not treated with reverse transcriptase.

FIG. 6

Transcription of US28 and the HCMV DNA polymerase gene in undifferentiated (untreated) and differentiated (PMA-treated) THP-1 cells. RT-PCR samples are visualized on agarose gels as described in Fig. 2. The primer sets that correspond to each of the panels are indicated on the right. The time points (p.i.) at which samples were taken for RT-PCR are shown at the top. d, days p.i.