Patchwork pattern of transcriptional reactivation in the lungs indicates sequential checkpoints in the transition from murine cytomegalovirus latency to recurrence - PubMed (original) (raw)

Patchwork pattern of transcriptional reactivation in the lungs indicates sequential checkpoints in the transition from murine cytomegalovirus latency to recurrence

S K Kurz et al. J Virol. 1999 Oct.

Abstract

The lungs are a significant organ site of murine cytomegalovirus (mCMV) latency. We have shown that activity of the major immediate-early promoter (MIEP), which drives the transcription from the ie1-ie3 transcription unit, does not inevitably initiate the productive cycle (S. K. Kurz, M. Rapp, H.-P. Steffens, N. K. A. Grzimek, S. Schmalz, and M. J. Reddehase, J. Virol. 73:482-494, 1999). Thus, even though MIEP activity governed by the MIEP-enhancer is unquestionably the first condition for recurrence, regulation of the enhancer by transcription factors is not the only mechanism controlling latency. Specifically, during latency, focal and stochastic MIEP activity in lung tissue was found to selectively generate IE1 transcripts, while transactivator-specifying IE3 transcripts were missing. This suggested a control of mCMV latency that is effectual at IE1-IE3 precursor mRNA cotranscriptional processing. Here we have used this model for studying the kinetics of reactivation and recurrence in individual lung tissue pieces after hematoablative, genotoxic treatment. Notably, reactivation was triggered, but the number of transcriptionally active foci in the lungs did not increase over time. This result is not compatible with a model of spontaneous reactivations accumulating after withdrawal of immune control. Instead, the data support the idea that reactivation is an induced event. In some pieces, focal reactivation generated IE3 transcripts but not gB transcripts, while other pieces contained foci that had proceeded to gB transcription, and only a few foci actually reached the state of virus recurrence. This finding indicates the existence of several sequentially ordered control points in the transition from mCMV latency to recurrence.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Transcriptional status of mCMV during latency in the lungs. A scheme illustrating the lobular anatomy of the lungs in ventral view is shown at the top. The lobes were cut into equal pieces listed on the lung map as #1 through #18. Minor size (weight) differences were compensated for by adjustment of the aliquots included in the assays. (Lower right) Verification of mCMV latency. The absence of infectious virus in the left lungs and postcaval lobes of mice LM1 to LM3 was demonstrated at 8 months after BMT and infection by testing the infectivity of lung tissue homogenates with the RT-PCR-based focus expansion assay (18). As a positive control, 0.05 PFU of purified mCMV was added to the homogenate of piece #10 before centrifugal infection of an MEF culture. Poly(A)+ RNA derived from this culture after 72 h of viral replication was serially diluted as indicated, whereas in the case of indicator cultures infected with homogenates of pieces #11 through #18, a constant amount of 100 ng of poly(A)+ RNA was subjected to ie1 exon 3-exon 4 (hereafter referred to as exon 3/4)-specific RT-PCR, yielding an amplification product of 188 bp. Shown are the autoradiographs obtained after gel electrophoresis, Southern blotting, and hybridization with the γ-32P-end-labeled oligonucleotide probe IE1-P (17), which is directed against the exon 3/4 splice junction. (Lower left) Random pattern of IE1-specific transcripts. Poly(A)+ RNA isolated directly from lung tissue pieces #1 through #9 (a 1/10 aliquot thereof [ca. 200 ng]) of mice LM1 to LM3 was subjected to the ie1 exon 3/4-specific RT-PCR. The signal obtained for LM3 piece #1 was reproduced with a second aliquot, and, throughout, pieces negative for IE1 transcripts remained negative when a second aliquot was tested. Testing of third and fourth aliquots of all preparations for IE3 transcripts and gB transcripts, respectively, gave negative results throughout. The presence of mRNA in the preparations that corresponded to negative pieces was verified for a fifth aliquot by RT-PCR specific for hypoxanthine phoshoribosyltransferase transcripts (not shown), as documented in detail previously (17).

FIG. 2

FIG. 2

Quantitation of the latent viral DNA in transcriptionally active and silent tissue pieces. In parallel with the isolation of the poly(A)+ RNA used for the transcriptional analyses shown in Fig. 1, DNA was isolated from lung tissue pieces #1 through #9 derived from mice LM1 to LM3. For each mouse, DNA pools corresponding to transcriptionally active pieces (pool a) and transcriptionally silent pieces (pool s) were formed, according to the pattern of IE1-specific transcription shown in Fig. 1, lower left. The DNA pools were titrated as indicated and were subjected to an ie1 exon 4-specific PCR. A negative control was provided by DNA isolated from uninfected lungs (Mock). As a standard for the quantitation, this mock DNA was supplemented with plasmid pIE111, titrated in duplicate, and subjected to PCR accordingly. (Top) Autoradiograph of the dot blot obtained after hybridization with a γ-32P-end-labeled internal oligonucleotide probe. (Bottom) Computed phosphorimaging data for the same blot. For the sake of clarity, the computations are depicted as graphs only for mouse LM2. Log-log plots of radioactivity (mean of duplicates in the case of the standard) measured as phosphostimulated luminescence (PSL) units (ordinate) versus the amount of sample DNA (abscissa) are shown. The upper line relates the amount of DNA to the number of plasmids in the pIE111 standard. Calculations were made from the linear portions of the graphs, as shown as an example for 300 ng of DNA containing 200 copies and 120 copies of viral DNA in the active and silent pools, respectively, of mouse LM2. The results, given as copies of the viral genome per 106 lung cells (6 μg of DNA) were as follows: for LM1, a = 1,320 and s = 1,480; for LM2, a = 4,000 and s = 2,400; and for LM3, a = 1,260 and s = 1,460.

FIG. 3

FIG. 3

Kinetics of mCMV recurrence after hematoablative treatment. Mice of the same cohort for which the establishment of latency was verified in Fig. 1 were subjected to gamma irradiation with a dose of 6.5 Gy. The recurrence of infectivity was monitored by the RT-PCR-based focus expansion assay on days 4, 8, and 12 after the treatment for lung tissue pieces #10 to #18 derived from the postcaval lobes and left lungs of mice RM1 through RM9. Shown are the autoradiographs obtained after hybridization of the amplification products with probe IE1-P (see the legend to Fig. 1). The incidence of recurrence is indicated for each time point.

FIG. 4

FIG. 4

Patterns of transcriptional reactivation. Transcripts of viral genes ie1, ie3, and gB were detected by respective RT-PCRs (17) for poly(A)+ RNAs derived from lung tissue pieces #1 through #9 (superior, middle, and inferior lobes) of mice RM1 through RM9, analyzed in groups of three on days 4, 8, and 12 after induction by gamma irradiation with a dose of 6.5 Gy. For standards to determine the sensitivity of detection, carrier poly(A)+ RNA derived from uninfected lung tissue was supplemented with a defined number of the respective in vitro-synthesized RNA molecules and titrated as indicated (top row). Throughout, RT-PCRs were performed with ca. 200 ng of sample poly(A)+ RNA, which represents 1/10 of the yield from one tissue piece. Faint or otherwise questionable signals (such as, e.g., IE1 in RM1 #4, IE1 in RM9 #6, and IE3 in RM7 #1) were either confirmed or rejected after testing of a second and, if necessary, a third aliquot. Note that some decisions need to be made from the original autoradiographs. Throughout, negative samples remained negative when further aliquots were tested. The reason for the apparently smaller size of the IE1 amplification product in RM1 #3 is under investigation. Shown are the autoradiographs obtained after hybridization with γ-32P-end-labeled probes IE1-P, IE3-P, and gB-P (for a map, see reference 17). Incidences of positive pieces are indicated for each time point.

FIG. 5

FIG. 5

Patchwork pattern of mCMV transcriptional reactivation and recurrence in the lungs. (Top) Pattern typical of latency. (Bottom) Pattern observed during reactivation and recurrence. The data shown as autoradiographs in Fig. 1, 3, and 4 are here compiled and illustrated as topographical maps of the lungs. Numbers 1 to 18 are assigned to individual tissue pieces. C, piece #10 was used as a positive control for the verification of latency (see Fig. 1, lower right). Pieces derived from superior, middle, and inferior lobes (#1 to #9) were tested for transcripts, and pieces derived from postcaval lobes and left lungs (ventral view) (#10 to #18) were tested for infectivity. Color code: uncolored, negative in the respective assays; red, positive for IE1 transcripts only; yellow, simultaneously positive for IE1 and IE3 transcripts; green, simultaneously positive for IE1, IE3, and gB transcripts; blue, Pieces containing recurrent virus. LM, latent mouse. RM, recurrent/reactivating mouse.

FIG. 6

FIG. 6

Images of focal activities during mCMV latency and reactivation: the orbital model. (Upper left) Prototypic latently infected lung. (Upper right) Prototypic reactivating/recurrently infected lung. Shown are topographical maps of the statistically generated prototypic lungs, with the number and type of foci indicated by symbols illustrating the sequential order of viral gene expression, which is ascending from IE1-specific transcription (red core) via IE3-specific cotranscriptional processing (yellow shell) and gB-specific transcription (green shell) to the production of infectious progeny virions (blue shell). (Box) Orbital model of mCMV reactivation, with sequentially ordered gene expression symbolized by colors that represent increasing “energy.” Checkpoints thought to be involved in the transition from latency to recurrence are indicated. The flash symbol represents the induction by gamma rays.

Similar articles

Cited by

References

    1. Angulo A, Messerle M, Koszinowski U H, Ghazal P. Enhancer requirement for murine cytomegalovirus growth and genetic complementation by the human cytomegalovirus enhancer. J Virol. 1998;72:8502–8509. - PMC - PubMed
    1. Balthesen M, Messerle M, Reddehase M J. Lungs are a major organ site of cytomegalovirus latency and recurrence. J Virol. 1993;67:5360–5366. - PMC - PubMed
    1. Dorsch-Häsler K, Keil G M, Weber F, Jasin M, Schaffner W, Koszinowski U H. A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. Proc Natl Acad Sci USA. 1985;82:8325–8329. - PMC - PubMed
    1. Grzimek N K A, Podlech J, Steffens H-P, Holtappels R, Schmalz S, Reddehase M J. In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus. J Virol. 1999;73:5043–5055. - PMC - PubMed
    1. Hermiston T W, Malone C L, Witte P R, Stinski M F. Identification and characterization of the human cytomegalovirus immediate-early region 2 that stimulates gene expression from an inducible promoter. J Virol. 1987;61:3214–3221. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources