Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo - PubMed (original) (raw)

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo

Seungmin Hwang et al. J Virol. 2008 Dec.

Abstract

Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.

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Figures

FIG. 1.

FIG. 1.

Construction of recombinant MHV-68 for bioluminescent imaging. (A) Schematic diagram of M3FL. Viral M3 promoter-driven firefly luciferase (FL) gene cassette was inserted between genomic coordinates 746 and 747 of MHV-68 WUMS (GenBank no. U97553). Nucleotide 1882 is the insertion site of the bacterial artificial chromosome vector sequence. TR, terminal repeat. (B) Genomic integrity of M3FL. BAC DNA from wild-type MHV-68 (WT) and M3FL was digested with EcoRI, HindIII, and SmaI and analyzed by agarose gel electrophoresis. Small white circles indicate the shift of digested fragment due to the insertion of the M3 promoter-driven FL gene cassette. The white triangle indicates the heterogenous fragment in the EcoRI-digested pattern, which includes the 40-bp repeat region. (C) Expression of FL from M3FL virus. The lysate of the cells infected with either WT or M3FL was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the expression of FL was analyzed by Western blotting using anti-FL (sc-57603; Santa Cruz). A Western blot using antibodies against viral structural proteins (ORF26 and M9) was included as a control for viral replication. (D) Multistep growth curve of wild-type MHV-68 and M3FL in NIH 3T12 cells. The data were compiled from three independent experiments, and standard deviations are shown as error bars. (E) In vitro correlation of FL activity with viral replication. NIH 3T12 cells were infected with M3FL at multiple MOIs, and the titer of infectious virus and the relative luciferase units (R.L.U.)/μg of protein in the whole-cell lysate were determined as described in Materials and Methods. (F) In vivo correlation of FL activity with viral replication. Mice were infected intranasally with 5 × 105 PFU of M3FL, and the titer of infectious virus and the number of RLU in the lungs of the infected mice were determined as described in Materials and Methods. (G) Acute replication of WT and M3FL in the lungs of the infected mice at 7 dpi. The titer of infectious virus and number of viral genome copies were determined by plaque assay (left panel) and quantitative real-time PCR (q-PCR) (right panel), respectively. (H) Latency establishment of WT and M3FL in the spleen at 14 dpi. The reactivatable latency and viral genome load were determined by infectious center (I.C.) assay (left panel) and quantitative PCR (right panel), respectively. (Left) The reactivatable latency as determined by an infectious center (I.C.) assay for 107 splenocytes (107 sp) is shown (four to six mice per group and per time point). Data are represented as means plus standard deviations (error bars). M3FL values that were not significantly different (n.s.) or were significantly different (P < 0.05 [ast]) from the value for the WT are indicated.

FIG. 2.

FIG. 2.

Spatial and temporal progression of MHV-68 infection via different routes of inoculation. Mice were infected intranasally (A), intraperitoneally (B), or orally (C) with 5 × 105 PFU of M3FL. Images at different time points are shown to represent acute infection in the primary site of infection, the spatiotemporal progression of viral replication, and the clearance and reappearance of viral replication. The day postinfection (e.g., D2 is day 2 postinfection) is shown at the bottom of each series of images.

FIG. 3.

FIG. 3.

Identification of novel sites of MHV-68 replication. (A) Ex vivo bioluminescent imaging showing the in situ localization of luciferase signal after intranasal infection. At the indicated day (D) postinfection in the figure, the infected mice were sacrificed and dissected to identify the signaling organs. Whether a particular organ showed signal (red plus signs) or not (black minus signs) at the given time is shown below the images. (B) Latency establishment of M3FL in the spleen, lung, salivary glands, thymus, and the subpopulations of cells in the thymus. Mice were infected with 5 × 105 PFU of M3FL, and the organs of the infected mice were harvested at 14 dpi. Reactivatable latency was measured by infectious center (I.C.) assay. (C) Restriction enzyme (EcoRI) digestion pattern of the reactivated viruses in different organs. The rightmost lane shows the EcoRI digestion pattern of BAC MHV-68.

FIG. 4.

FIG. 4.

Investigation of M3FL infection in different sites at multiple time points. (A) Comparable replication of M3FL among mice after intranasal infection with 5 × 105 PFU of M3FL. Images depicted represent acute infection in the nose and the lung (day 6 postinfection [D6]), the transition of replication from the lung to the spleen (D14), and the reappearance of viral replication (D45). (B and C) A region of interest was manually selected over the signals, and the intensity was analyzed using LivingImage software and expressed as photon flux (photons/s/cm2/steradian). As representative data, the comparison of bioluminescence signals among the four mice (B) and the spatiotemporal progression of the signals in a single mouse (C) are shown. For the reliable comparison of signal intensities, the maximum photon flux values were measured from each ROI and shown here.

FIG. 5.

FIG. 5.

In vivo reactivation of MHV-68 from latency. When there was no significant bioluminescent signal in the whole body of infected mice, the mice were intraperitoneally treated with 0.3 mg of Velcade/kg (at ca. 150 dpi) (A) or 50 mg/kg of cyclosporine A (at ca. 90 dpi) (B) on the days indicated in red (D147, 147 dpi) and continuously monitored. (C) Total RNAs were extracted from the infected NIH 3T12 cells (3 dpi; MOI of 0.05) (lane 1), uninfected NIH 3T12 cells (lane 2), the spleen of the infected mouse (ca. 100 dpi) (lane 3), or the spleen of the infected mouse (ca. 100 dpi) after CspA treatment (50 mg/kg, twice) (lane 4). cDNAs were prepared, and the transcript levels of ORF50 (immediate-early), ORF57 (early), ORF29 (late), and ORF73 (latent) were detected by PCR. D, days postinfection.

FIG. 6.

FIG. 6.

Different reactivation of M3FL via different routes of infection. (A) In vivo reactivation of M3FL a long time after infection via different routes. All the long-term infected mice were intraperitoneally treated with 50 mg of CspA/kg twice (every third day). Before and 6 days after initial administration of CspA, the mice were imaged. ID, identification number. (B) Ex vivo bioluminescent imaging showing the in situ localization of luciferase signal from M3FL a long time after infection via different routes. Whether a particular organ showed signal (red plus sign) or not (green minus sign) at the given time is shown below the photographs.

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