U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture

Abstract

Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. Evidence of ability to establish latent infection has not been available, but by analogy with other herpesviruses it could be expected that IE genes that regulate and transactivate late genes would not be expressed. We report that peripheral blood mononuclear cells of healthy individuals infected with HHV-6 express the U94 gene, transcribed under IE conditions. Transcription of other IE genes (U16/17, U39, U42, U81, U89/90, U91) was not detected. To verify that U94 may play a role in the maintenance of the latent state, we derived lymphoid cell lines that stably expressed U94. HHV-6 was able to infect these cells, but viral replication was restricted. No cytopathic effect developed. Furthermore, viral transcripts were present in the first days postinfection and declined thereafter. A similar decline in the level of intracellular viral DNA also was observed. These findings are consistent with the hypothesis that the U94 gene product of HHV-6 regulates viral gene expression and enables the establishment and/or maintenance of latent infection in lymphoid cells.

Figure 1

Schematic representation of the HHV-6 genome, showing the physical location of the ORFs analyzed in this study. Arrows indicate the direction of transcription. U16/17, U91, and U89/90 correspond to spliced transcripts.

Figure 2

(A) Southern Blot of DNA extracted from Rep-transformed cell lines and hybridized to radiolabeled U94 DNA probe. The numbers 10, 1, and 0.1 indicate lanes containing, respectively, 10, 1, and 0.1 copy equivalents of U94 DNA. Rep-expressing lines RepCl2, RepCl3, and RepCl4 are shown in lanes A, B, and C, while the control cell line, N1, and parental JJhan cells are shown in lanes D and E. DNA was digested with HindIII to excise U94 from the vector as a 1.5-kb band. (B) Presence of U94 transcripts in Rep-expressing cell lines (2, 3, 4). RNA (no initial reverse transcription step) and cDNA (+RT) were amplified by PCR and analyzed by agarose gel. Lane C represents a control amplification reaction, performed by using 10,000 DNA molecules of U94 template.

Figure 3

Semiquantitative analysis of viral DNA in cell lines infected with cell-free HHV-6. DNA was extracted from Rep-transformed cell lines (Cl2, Cl3, Cl4), parental cells (JJ), and N1 control cell line at various time points after viral infection [either immediately postinfection (0), or at 7, 22, and 28 days postinfection]. Serial 10-fold dilutions of cellular DNA (starting at 0.1 μg) were analyzed by PCR, using primers specific for the viral U31 ORF. The arrows point to the highest dilution of template DNA yielding a positive signal upon PCR amplification.

Figure 4

Analysis of HHV-6 transcription in Rep-expressing cell lines infected with cell-free viral inoculum. RNA extracted from Rep-expressing cell lines 2, 3, and 4 and from parental JJahn cells (JJ) and control N1 cells was analyzed by RT-PCR, using primers specific for the viral ORFs U42, U91, and U16/17. U91 and U16/17 are spliced mRNAs. The arrow points to a faint band that reproduced poorly in the print.

Figure 5

Results of a reconstruction experiment, comparing the sensitivities of the various PCRs. DNA was extracted from HHV-6-infected cells, and serial 10-fold dilutions were analyzed by nested PCR, using primers specific for the viral ORFs U16/17, U42, and U94. The size of the PCR products produced is expressed in base pairs.

Figure 6

Presence of U94 mRNA in PBMCs from healthy, HHV-6-positive, adult blood donors. RNA was extracted from PBMCs from healthy individuals harboring HHV-6 DNA sequences and analyzed by RT-PCR by using primers specific for the U94 ORF (lanes C–F and I–N). Control reactions (to test for possible DNA contamination of RNA specimens and/or contamination of PCRs) are shown in lanes A and G (extraction blanks) and B and H (blank reactions).