Characterization of an antisense transcript spanning the UL81-82 locus of human cytomegalovirus

Abstract

In this study we present the characterization of a novel transcript, UL81-82ast, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR, UL81-82ast was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found. UL81-82ast was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.

FIG. 1.

Graphic representation of the HCMV genome, relative positions of genes analyzed in this study, and primers and probes used.

FIG. 2.

cDNA library construction from monocyte mRNA. (A) Presence of HCMV DNA in the donor's monocytes. DNA was extracted from the isolated monocytes and tested for the presence of HCMV DNA by nested PCR, using primers IEP 4AII and 2BII, for the first round, and IEP 3A and 3B, for the second round. A positive (DNA from HCMV-infected cells) and a negative control are included. (B) Absence of IE-1 transcripts in monocyte RNA. Total RNA and poly(A)⁺ RNA samples were subject to cDNA synthesis using random primers. As a positive control, DNA extracted from HCMV-infected HFF cells was used. All samples were subject to nested PCR using HCMV lytic genes primers IEP 4AII and 2BII, for the first round, and IEP 3A and 3B, for the second round. To test the quality of the cDNA/DNA samples, PCR was performed using β-actin primers (C) Size distribution of cDNA inserts in the phage DNA library. Phage DNA from 10 randomly selected clear plaques on NZY agar plates containing X-Gal and IPTG was amplified by using primers flanking the cDNA insert. PCR products representing cDNA inserts from approximately 500 bp to >3 kb were resolved by agarose gel electrophoresis and photographed after ethidium bromide staining. Bands from lanes 1 to 7 (marked with arrows) were too weak to be observed in the picture.

FIG. 3.

Presence of HCMV transcripts in the phage DNA. (A) Presence of previously described CMV latency-associated transcripts. The presence of CMV latency-associated transcripts was studied in the library DNA as well as in the initial total RNA 3 extraction. DNA from the library and cDNA from total RNA 3 were tested by PCR using primer combinations antiCLT F1-R1, CLT F1-R2, and CLT F2-R2 (lanes 1 to 3, respectivley), as well as other combinations (data not shown). DNA from the library was tested by PCR using HCMV UL81-specific primers. As a positive control, DNA extracted from HCMV-infected HFF cells was used. (B) PCR using primers 81F1 and R1 or primers 81F2 and R1. The bands from the library sample are marked with an arrow. (C) Heminested PCR. To obtain higher amplification a heminested PCR was performed using primer 81F1-R1 in the first round and 81F2-R1 in the second round.

FIG. 4.

Orientation of the UL81-82 transcript present in the library. (A) Directional cloning of cDNAs into phage library allows differentiating direction of the transcripts. (B) PCR using HCMV-specific primers from extracted library DNA. For lanes 1 to 16, odd numbers are library DNA, and even numbers are water controls. Positive controls in lanes 17 and 18, heminested amplification of library DNA with primers 81F2-81R2 (first round with 82F1-81R1). Primers used: lanes 1 and 2, 82Fi1-ZapF; lanes 3 and 4, 82F1-ZapF; lanes 5 and 6, 82F2-ZapF; lanes 7 and 8, 81F1-ZapF; lanes 9 and 10, 82Fi1-ZapR; lanes 11 and 12, 82F1-ZapR; lanes 13 and 14, 82F2-ZapR; lanes 15 and 16, 81F1-ZapR. Southern blotting and hybridization using PCR with primers UL81F2 and UL81R3 and HCMV DNA as a template (lanes 17 and 18) for the probe preparation. Developed for 15 minutes and 1 hour. (C) PCR using UL82 region and lambda vector primers from extracted library DNA. PCR using primer 82F1-ZapF or primer 82F1-ZapR was performed. The bands labeled a, b, and c were sequenced. (D) Orientation of the transcript present in monocyte RNA of latently infected patient using 3′RACE. Samples labeled 82Fs-AP underwent cDNA synthesis using primer UL81F1, and nested PCR using primers UL81F1-AP and UL81F2-UAP. Samples labeled 82Rs-AP underwent cDNA synthesis using primer UL81R1, and nested PCR using primers UL81R1-AP and UL81R2-UAP. mRNA corresponds to the poly(A) purification sample used for the library, and the sample lytic RNA is RNA extracted from HFF cells infected with Smith virus under lytic conditions. AP = adaptor primer, UAP = universal adaptor primer.

FIG. 5.

Presence of UL82ast in bone marrow from latently infected patients. RNA was extracted by the Trizol method from latently HCMV-infected bone marrow samples. cDNA synthesis using random primers was performed using the extracted RNAs. Nested-PCR amplifications were performed using primers UL82F1 and UL81R1 in the first amplification and UL82F2 and UL81R2 in the second. Lanes 1 to 3 represent HCMV-negative donors 1051, 989, and 1134; lanes 4 to 8 represent the cDNAs from HCMV-positive donors 535, 553, 638, 234, and 376; lane 9 is the negative water control. Southern blotting and hybridization, developed for 15 min. Same probe as in Fig. 3B (PCR using primers UL81F2 and UL81R3 and HCMV DNA as a template).

FIG. 6.

Analysis of UL81-82ast and its protein product. (A) Map of the area showing the transcript (underlined) and the UL81-82ast ORF. *, Relative position compared to strain AD169 (accession number X17403). (B) Alignment of UL82as protein from several CMV genomes. Protein alignment was done using MegaAlign, from Lasergene (DNAstar, Inc.), and Clustal W algorithms. The compared genomes included human herpesvirus 5 strain AD169, Merlin strain, FIX-BAC isolate, TR-BAC isolate, PH-BAC isolate, Towne-BAC isolate, Toledo isolate, and chimpanzee CMV. See accession numbers in the Materials and Methods section.

FIG. 7.

Presence of UL82as protein in HCMV-infected cells. (A) Western blot of 12% acrylamide gel developed using rabbit polyclonal serum anti-UL82as as a primary antibody. Lane 1, E. coli purified recombinant UL82as protein. Lane 2, uninfected HFF cells. Lanes 3 to 5, HCMV clinical isolate-infected HFF cells, 7 hours postinfection, 24 hours postinfection, and 6 days postinfection, respectively. The molecular marker lane shows a 16-kDa band. (B) Western blot of 12% acrylamide gel developed using anti-IE1/IE2 monoclonal antibody mAb810 as a primary antibody. Lane 1, uninfected HFF cells. Lanes 2 to 4, HCMV clinical isolate-infected HFF cells, 7 hours postinfection, 24 hours postinfection, and 96 hours postinfection, respectively.

FIG. 8.

Presence of UL82as protein in HCMV-infected HFF cells. Cells were stained using monoclonal anti-IE1/2 (Mab810), and rabbit polyclonal anti-UL82as as primary antibodies and anti-mouse Alexa Fluor 555-conjugated (imaged red) and anti-rabbit Alexa Fluor 488 (imaged green)-conjugated as the secondary antibody. Hoechst stain was used to identify cell nuclei, which are shown in blue. (A) Indirect immunofluorescence of HCMV-infected cells at different time points after infection (8, 24, and 96 hours postinfection, respectively). Magnification, 100×. (B) Confocal microscopy of HCMV-infected cells at different time points after infection (4, 24, and 96 hours postinfection, respectively). Magnification, 40×. (C) Apparent presence of UL82as in nucleoli. Visualized by indirect immunofluorescence and confocal microscopy after 24 hours postinfection in HCMV-infected HFF.