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. 1999 Jan;73(1):482-94.
doi: 10.1128/JVI.73.1.482-494.1999.

Focal transcriptional activity of murine cytomegalovirus during latency in the lungs

S K Kurz  1 , M Rapp, H P Steffens, N K Grzimek, S Schmalz, M J Reddehase
Affiliations

Focal transcriptional activity of murine cytomegalovirus during latency in the lungs

S K Kurz et al. J Virol. 1999 Jan.

Abstract

Interstitial pneumonia is a frequent and critical manifestation of human cytomegalovirus (CMV) disease in immunocompromised patients, in particular in recipients of bone marrow transplantation. Previous work in the murine CMV infection model has identified the lungs as a major organ site of CMV latency and recurrence. It was open to question whether the viral genome is transcriptionally silent or active during latency. Transcription could be latency associated and thus be part of the latency phenotype. Alternatively, transcriptional activity could reflect episodes of reactivation. We demonstrate here that transcription of the immediate-early (IE) transcription unit ie1-ie3 selectively generates ie1-specific transcripts during latency. Notably, while the latent viral DNA was found to be evenly distributed in the lungs, transcription was focal and randomly distributed. This finding indicates that IE transcription is not a feature inherent to murine CMV latency but rather reflects foci of primordial reactivation. However, this reactivation did not initiate productive infection, since ie3 gene mRNA specifying the essential transactivator IE3 of murine CMV early gene expression was not detectable. Accordingly, transcripts encoding gB were absent during latency. By contrast, during induced virus recurrence, IE-phase transcription switched from focal to generalized and ie3-specific transcripts were generated. These data imply that latency and recurrence are regulated not only at the IE promoter-enhancer and that there exists an additional checkpoint at the level of precursor RNA splicing. We propose that focal transcription reflects random episodes of nonproductive reactivation that get terminated before IE3 is expressed and ignites the productive cycle.

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Figures

FIG. 1
FIG. 1
Map of the transcriptional analysis. The HindIII physical map of the murine CMV Smith strain genome is shown in the center. The gB gene is expanded below. The ie1-ie3 transcription unit (IE1/3) and the IE1 and IE3 splicing products derived thereof are resolved to greater detail at the top. IE1, IE3, and gB are drawn to scale. Boxes represent the genomic organization with exons (Ex) indicated. Ex1 is noncoding. The wavy lines illustrate the positions and lengths of in vitro-synthesized RNAs. The asterisk marks the 5′ transcriptional start site of the ie1-ie3 transcription unit. Locations of primers and probes are illustrated by black bars at the amplified sequences. The suffix -RT denotes a specific primer for reverse transcription. An oligo(dT) primer was used in the case of gB. The suffix -R denotes a reverse primer, -F denotes a forward primer, and -P denotes a hybridization probe. MIEPE, major IE promoter-enhancer.
FIG. 2
FIG. 2
Kinetics of viral DNA clearance in hematopoietic cells and their progeny. Sex-mismatched BMT was performed with BMC derived from male (XY) BALB/c donors transplanted into female (XX) BALB/c recipients. DNA was isolated, at the indicated time points after BMT and infection, from blood and BMC pooled from three recipients per time point. (A) Quantitation of viral DNA in blood. (B) Quantitation of viral DNA in BM. On the left are shown cellular gene controls documenting equivalent efficacy of PCR for DNA preparations derived from blood and BM at 12 months after BMT and infection. The cellular sequence amplified was that from the tdy gene, which is located on the Y chromosome of the repopulating, donor-derived male cells. DNA isolated from uninfected female recipients of a sex-matched syngeneic BMT (XX-DNA/mock DNA) was used for the negative controls. Amplification of a 363-bp sequence within exon 4 of gene ie1 of murine CMV is shown on the right. Mock DNA derived from blood or BM of the uninfected female BMT recipients was supplemented with plasmid pIE111, which includes ie1, and was titrated in duplicate as a standard for the quantitation. Shown are the autoradiographs of the dot blots obtained after hybridization with the respective γ-32P-end-labeled internal oligonucleotide probes. (C) Comparison of the viral DNA loads in blood and BM. The radioactivity per dot was measured by phosphorimaging, and the number of viral ie1 genes per 600 ng of DNA, which is equivalent to the DNA content of 105 diploid cells, was calculated from the linear portions of the titrations. The arrow marks the time point for all subsequent analyses.
FIG. 3
FIG. 3
General outline of the approach. A scheme illustrating the lobular anatomy of the lungs in ventral view is shown at the upper right. To examine the results in their topographical relation, the lobes were cut into equal pieces designated on the lung map as #1 through #18. Minor size (weight) differences were compensated by adjustment of the aliquots included in the assays. (A) Verification of murine CMV latency. The absence of infectious virus was confirmed at 12 months after BMT and infection for latent mouse LM1 by using a high-sensitivity assay of infectivity, the RT PCR-based focus expansion assay. As a positive control, 0.05 PFU of purified murine CMV were added to the homogenate of piece #10 before the infection of an indicator culture of permissive mouse fetal cells. Poly(A)+ RNA derived from this culture after 72 h of viral replication was serially diluted as indicated, whereas for indicator cultures infected with homogenates of pieces #11 through #18 a constant amount (100 ng) of poly(A)+ RNA was subjected to an ie1 exon 3/4-specific RT PCR. Shown are the autoradiographs obtained after gel electrophoresis, Southern blotting, and hybridization with the γ-32P-end-labeled oligonucleotide probe IE1-P directed against the exon 3/4 splice junction. For the indicator culture corresponding to piece #18, the presence of RNA in the preparation is exemplarily demonstrated by RT PCR specific for the cellular housekeeping gene transcript HPRT. (B) Direct analysis of in vivo transcriptional activity. Poly(A)+ RNA isolated from pieces #1 through #9 (a 1/10 aliquot thereof [200 ng]) was subjected to the ie1 exon 3/4-specific RT PCR. As a positive control and standard, carrier poly(A)+ RNA derived from uninfected lung tissue was supplemented with the indicated numbers of in vitro-synthesized IE1 RNA molecules. HPRT-specific RT PCR served to document the presence of RNA in all samples. (C) For increasing the sensitivity of detection, pieces scored as negative in the analysis illustrated in panel B were retested by including a 7/10 aliquot of the poly(A)+ RNA yield in the ie1 exon 3/ 4-specific RT PCR.
FIG. 4
FIG. 4
Determination of the viral DNA load during murine CMV latency in the lungs. In parallel with the isolation of the poly(A)+ RNA used for the transcriptional analyses shown in Fig. 3B and C, DNA was isolated from lung tissue pieces #1 through #9 derived from mouse LM1. The DNA from the individual pieces as well as a pool of the DNA from all nine pieces (Pool) were titrated and subjected to an ie1 exon 4-specific PCR. A negative control (Mock) was provided by DNA derived from uninfected lungs. As a standard for the quantitation, the mock DNA was supplemented with plasmid pIE111. (Top) Autoradiograph of the dot blot obtained after hybridization with a γ-32P-end-labeled internal oligonucleotide probe. (Bottom) Computed phosphorimaging results for the same blot. For the sake of clarity, the computations are depicted only for the pool, representing the average of all samples, as well as for the individual samples with the lowest load (Min) and the highest load (Max) among the nine samples tested. Log-log plots of radioactivity measured as phosphostimulated luminescence (PSL) units (ordinate) versus the amount of sample DNA (abscissa) are shown. The upper rule relates the amount of DNA to the number of plasmids in the pIE111 standard.
FIG. 5
FIG. 5
Stochastic distribution of IE1 transcripts in latently infected lungs. Transcription from the ie1 gene was analyzed for mice LM1 through LM5 as outlined in Fig. 3B for mouse LM1. The analysis for mouse LM1 was repeated (LM1*) with the final aliquot of the poly(A)+ RNA preparation. The locations of positive and negative pieces in the three lobes of the lungs tested are illustrated by topographical maps, with transcriptionally active pieces being indicated by shading. Samples marked by one, two, and three asterisks were classified as samples with low, intermediate, and high transcriptional activity, respectively, and were used later for the quantitation of transcripts (see Fig. 7).
FIG. 6
FIG. 6
Estimation of the average number of transcriptional foci in latently infected lungs. The statistical calculations were based on the patterns of transcription observed for 45 pieces derived from the superior, middle, and inferior lung lobes of five latently infected mice (see Fig. 5). The observed fraction, f(0), of transcriptionally negative lung pieces was used to calculate the Poisson distribution parameter λ (lambda). Based on λ, the fraction of pieces containing n (n = 0, 1, 2, and 3) foci, F(n), was calculated, and the results were extrapolated to a prototypic complete lung. The upper estimate was obtained by assuming that neighboring positive pieces are always both transcriptionally active. In that case, f(0) = 21/45. The lower estimate was obtained by assuming that neighboring positive pieces always share a transcriptional focus and count as a single positive piece. In that case, f(0) = 29/45. Shown are topographical maps of the statistically generated prototypic latent lungs, with pieces containing n (n = 0, 1, 2, and 3) foci being indicated by the corresponding number of dots.
FIG. 7
FIG. 7
Quantitation of IE1 transcripts in transcriptionally active lung tissue pieces. Positive pieces from mice LM1 through LM5 (see Fig. 5) were classified as having low, intermediate, or high transcriptional activity. Representative samples of the three so defined classes (indexed in Fig. 5 by the number of asterisks) were used to form poly(A)+ RNA pools 1, 2, and 3, respectively. Quantitation was performed by titration of the RNA pools, ie1 exon 3/ 4-specific RT PCR, and phosphorimaging. Shown are the autoradiographs. In vitro-synthesized IE1 transcripts provided the standard. The results are expressed as rounded-off numbers of IE1 transcripts per piece.
FIG. 8
FIG. 8
Survey of productive cycle transcription in latently infected lungs. Transcription from genes ie1 (exon 3/4 of the ie1-ie3 transcription unit), ie3 (exon 3/5 of the ie1-ie3 transcription unit), and gB was analyzed for the high-activity poly(A)+ RNA pool (pool 3; see Fig. 7) by the appropriate RT PCRs. (Top) Ethidium bromide-stained gels verifying the correct sizes of the amplificates. M, lane M contains the 100-bp size markers; ØRT, control reactions performed with 200-ng samples and all components except the RT. (Bottom) Corresponding Southern blot autoradiographs obtained after hybridization with the γ-32P-end-labeled oligonucleotide probes. For the IE1 and IE3 transcripts, the probes were directed against the exon 3-exon 4 (probe IE1-P) and exon 3-exon 5 (probe IE3-P) splicing junctions, respectively.
FIG. 9
FIG. 9
Analysis of reactivation and recurrent infection in the lungs. Recurrence of infectious with virus from latently infected lungs was induced at 12 months after BMT and infection by total-body γ-irradiation with a dose of 6.5 Gy. Data are shown for a particular case on day 14 after the irradiation. (A) Assay of infectivity. Infectious virus was detected in lung tissue pieces #10 through #18 (left lungs and postcaval lobe) by using the RT PCR-based focus expansion assay (details are as described in the legend for Fig. 3). (B) Detection of IE1 and IE3 transcripts. Poly(A)+ RNA was isolated from lung tissue pieces #1 through #9 (remaining three lobes) on day 14 after the irradiation, and 1/10 aliquots (ca. 200 ng) were subjected to the RT PCRs.
FIG. 10
FIG. 10
Quantitative comparison between IE1 transcripts and IE3 transcripts during recurrence. Aliquots of the poly(A)+ RNAs derived from lung tissue pieces #1 through #9 (see Fig. 9) were pooled. Aliquots of the pool (200 ng, which equals 1/10 of the yield of one piece) were serially diluted and subjected to the RT PCRs. The in vitro-synthesized transcripts were titrated in parallel as standards for the quantitation. (Top) Autoradiographs obtained after gel electrophoresis of the amplificates, Southern blotting, and hybridization with the γ-32P-end-labeled oligonucleotide probe IE1/3-P directed against the common exon 3. (Bottom) Computed phosphorimaging data for the same blots. Log-log plots of radioactivity measured as phosphostimulated luminescence (PSL) units (ordinate) versus the amount of poly(A)+ RNA (abscissa) are shown. The upper rule relates the amount of poly(A)+ RNA to the number of molecules in the standard.
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