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Comparative Study
. 2001 Jun;75(11):4955-63.
doi: 10.1128/JVI.75.11.4955-4963.2001.

Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice

R Rochford  1 , M L Lutzke, R S Alfinito, A Clavo, R D Cardin
Affiliations
Comparative Study

Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice

R Rochford et al. J Virol. 2001 Jun.

Abstract

A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.

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Figures

FIG. 1
FIG. 1
(A) MHV-68 gene expression in vitro. 3T3 cells were infected with MHV-68 at an MOI of 5. Cells were harvested at 8 hpi, RNA was extracted, and 5 μg of total RNA was subjected to RPA analysis using the γ-3 or γ-4 riboprobe templates. The protected RNA fragments were visualized with a PhosphorImager. RNAs from two separate experiments were analyzed (lanes 1 and 2). (B) The PI counts for each protected probe fragment were obtained, and the data are presented as a percentage of the internal housekeeping (i.e., L32) signal present in each lane. The average values from two experiments are presented.
FIG. 2
FIG. 2
Assessment of the sensitivity of the RPA. Total RNA was extracted from OMK cells at 24 hpi, serially diluted, and analyzed with the γ-3 probe set. (A) Shown is a phosphorimage following exposure of the dried gel to a PhosphorImager screen. (B) The PhosphorImager was used to quantify the signal present in the Rta and M3 protected bands of the dried gel. Log-log plots are shown for PI counts versus input total RNA amounts.
FIG. 3
FIG. 3
Determination of kinetic class of MHV-68 transcripts. 3T3 cells were infected with MHV-68 in the presence of CHX or PAA or were left untreated. Total RNA was harvested at 8 hpi (CHX treated) or 24 hpi (PAA treated and untreated), and 2.5 μg of total RNA was analyzed by RPA using the γ-3 probe set (A) or γ-4 probe set (B). Shown are phosphorimages following exposure of the dried gel to a PhosphorImager screen.
FIG. 4
FIG. 4
Time course of MHV-68 transcript expression in vitro. 3T3 cells were infected with MHV-68 in duplicate, RNA was harvested at 1, 2, 4, 8, and 24 hpi, and 2.5 μg of total RNA was analyzed for expression of MHV-68 transcripts by RPA with the γ-3 probe set (A) or γ-4 probe set (B). Shown are phosphorimages following exposure of the dried gel to a PhosphorImager screen. The upper ranges in PI value were 1,000 (A, left panel) and 25 (A, right panel; B).
FIG. 5
FIG. 5
Comparison of MHV-68 transcript levels following in vivo infection. RNA extracted from lung (3 dpi), spleen (10 dpi), and MLN (10 dpi) was analyzed by RPA using the γ-3 probe set or γ-4 probe set. PI counts were obtained for each protected probe fragment, and the data are presented as a percentage of the L32 signal. Three separate organs were analyzed for both lung and spleen samples, while two organs were assessed for MLN. The average value is shown.
FIG. 6
FIG. 6
Kinetics of expression of the candidate latency-associated transcripts following in vivo infection. RNA, extracted from lung, spleen, and MLN for days 1 through 10 postinfection and every other day until 16 dpi, was subjected to RPA analysis using the γ-3 or γ-4 riboprobe templates. PI counts were obtained for the protected probe fragments for the candidate latency-associated transcripts (K3, M3, M9, M8, ORF74, OR73, and M11), and the data are presented as a percentage of the L32 signal.
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References

    1. Cardin R D, Brooks J W, Sarawar S R, Doherty P C. Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells. J Exp Med. 1996;184:863–871. - PMC - PubMed
    1. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. - PubMed
    1. Clambey E T, Virgin IV H W, Speck S H. Disruption of the murine gammaherpesvirus 68 M1 open reading frame leads to enhanced reactivation from latency. J Virol. 2000;74:1973–1984. - PMC - PubMed
    1. Flano E, Husain S M, Sample J T, Woodland D L, Blackman M A. Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages. J Immunol. 2000;165:1074–1081. - PubMed
    1. Hobbs M V, Weigle W O, Noonan D J, Torbett B E, McEvilly R J, Koch R J, Cardenas G J, Ernst D N. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol. 1993;150:3602–3614. - PubMed

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