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Measles virus induces persistent infection by autoregulation of viral replication

View Article: PubMed Central - PubMed

ABSTRACT

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

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Establishment of cell lines with persistent MV infection.B95a cells were infected with rMV in (a–e). (a) Cellular images of B95a cells, uninfected or at 4 days, 2 weeks, or 2 months post-MV infection. (b) Growth curve of B95a cells during the establishment phase of MV persistent infection. MV-infected B95a cells were counted every 5 days from 17 dpi. Control uninfected cells were also counted from same day. Error bars are SD of three independent infections. (c) Cell death of MV-infected B95a cells. Genomic DNA content was analysed by propidium iodide staining. The percentages of sub-G1 populations are graphed as dead cells. (d) Intracellular MV N protein staining of persistently MV-infected B95a cells. Filled and open histograms indicate uninfected and persistently MV-infected B95a cells, respectively. (e) Cell-free virion release from acute and persistent MV infection. For acute infection, B95a cells were infected with rMV-HL for 1 h at a MOI of 0.1 followed by culture medium exchange. Concurrently, equal numbers of persistently rMV-infected B95a cells were cultured, and 3 days later, the titres of three independent acute infections and three independently established persistently MV-infected cells were determined and graphed. Horizontal bars indicate averages. The cellular image shows the level of syncytia formation induced by virus released from persistently MV-infected cells. (f) B95a cells were infected with rMV-EGFP as described above. EGFP fluorescence was analysed by flow cytometry at the indicated times post-infection. Markers indicate the range of the EGFP-negative population.
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f1: Establishment of cell lines with persistent MV infection.B95a cells were infected with rMV in (a–e). (a) Cellular images of B95a cells, uninfected or at 4 days, 2 weeks, or 2 months post-MV infection. (b) Growth curve of B95a cells during the establishment phase of MV persistent infection. MV-infected B95a cells were counted every 5 days from 17 dpi. Control uninfected cells were also counted from same day. Error bars are SD of three independent infections. (c) Cell death of MV-infected B95a cells. Genomic DNA content was analysed by propidium iodide staining. The percentages of sub-G1 populations are graphed as dead cells. (d) Intracellular MV N protein staining of persistently MV-infected B95a cells. Filled and open histograms indicate uninfected and persistently MV-infected B95a cells, respectively. (e) Cell-free virion release from acute and persistent MV infection. For acute infection, B95a cells were infected with rMV-HL for 1 h at a MOI of 0.1 followed by culture medium exchange. Concurrently, equal numbers of persistently rMV-infected B95a cells were cultured, and 3 days later, the titres of three independent acute infections and three independently established persistently MV-infected cells were determined and graphed. Horizontal bars indicate averages. The cellular image shows the level of syncytia formation induced by virus released from persistently MV-infected cells. (f) B95a cells were infected with rMV-EGFP as described above. EGFP fluorescence was analysed by flow cytometry at the indicated times post-infection. Markers indicate the range of the EGFP-negative population.

Mentions: To determine whether or not mutations in the viral genome are dispensable for the establishment of persistent MV infection, we analysed cells and viruses during the early phase of persistent MV infection in cultured cells. According to previous studies, MV establishes persistent infection in virtually any cell type121314. Thus, we used B95a cells, which are marmoset lymphoblastoid cells, to investigate the mechanism of persistent infection because these cells are highly susceptible to MV infection and can easily achieve 100% infection during persistent infection. In the early phase of MV infection (4 days post-infection [dpi]), most of the cells formed syncytia and died (Fig. 1a). The few remaining cells, however, started to grow again (Fig. 1a). To determine when all of the cells in culture had established a persistent infection, we monitored cellular growth and cell death during the course of MV infection (Fig. 1b and c). Although syncytium formation and cell death faded out at 2 weeks post-infection (Fig. 1a and c), cell growth was severely impaired until 28 dpi when cells started to grow in a manner similar to that of uninfected controls (Fig. 1b). A second crisis occurred between 23 and 28 dpi in all three cultures independently, prior to the re-establishment of cell growth (Fig. 1c). After the establishment of persistent infection, the cell morphology was indistinguishable from that of uninfected cells (Fig. 1a).


Measles virus induces persistent infection by autoregulation of viral replication
Establishment of cell lines with persistent MV infection.B95a cells were infected with rMV in (a–e). (a) Cellular images of B95a cells, uninfected or at 4 days, 2 weeks, or 2 months post-MV infection. (b) Growth curve of B95a cells during the establishment phase of MV persistent infection. MV-infected B95a cells were counted every 5 days from 17 dpi. Control uninfected cells were also counted from same day. Error bars are SD of three independent infections. (c) Cell death of MV-infected B95a cells. Genomic DNA content was analysed by propidium iodide staining. The percentages of sub-G1 populations are graphed as dead cells. (d) Intracellular MV N protein staining of persistently MV-infected B95a cells. Filled and open histograms indicate uninfected and persistently MV-infected B95a cells, respectively. (e) Cell-free virion release from acute and persistent MV infection. For acute infection, B95a cells were infected with rMV-HL for 1 h at a MOI of 0.1 followed by culture medium exchange. Concurrently, equal numbers of persistently rMV-infected B95a cells were cultured, and 3 days later, the titres of three independent acute infections and three independently established persistently MV-infected cells were determined and graphed. Horizontal bars indicate averages. The cellular image shows the level of syncytia formation induced by virus released from persistently MV-infected cells. (f) B95a cells were infected with rMV-EGFP as described above. EGFP fluorescence was analysed by flow cytometry at the indicated times post-infection. Markers indicate the range of the EGFP-negative population.
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f1: Establishment of cell lines with persistent MV infection.B95a cells were infected with rMV in (a–e). (a) Cellular images of B95a cells, uninfected or at 4 days, 2 weeks, or 2 months post-MV infection. (b) Growth curve of B95a cells during the establishment phase of MV persistent infection. MV-infected B95a cells were counted every 5 days from 17 dpi. Control uninfected cells were also counted from same day. Error bars are SD of three independent infections. (c) Cell death of MV-infected B95a cells. Genomic DNA content was analysed by propidium iodide staining. The percentages of sub-G1 populations are graphed as dead cells. (d) Intracellular MV N protein staining of persistently MV-infected B95a cells. Filled and open histograms indicate uninfected and persistently MV-infected B95a cells, respectively. (e) Cell-free virion release from acute and persistent MV infection. For acute infection, B95a cells were infected with rMV-HL for 1 h at a MOI of 0.1 followed by culture medium exchange. Concurrently, equal numbers of persistently rMV-infected B95a cells were cultured, and 3 days later, the titres of three independent acute infections and three independently established persistently MV-infected cells were determined and graphed. Horizontal bars indicate averages. The cellular image shows the level of syncytia formation induced by virus released from persistently MV-infected cells. (f) B95a cells were infected with rMV-EGFP as described above. EGFP fluorescence was analysed by flow cytometry at the indicated times post-infection. Markers indicate the range of the EGFP-negative population.
Mentions: To determine whether or not mutations in the viral genome are dispensable for the establishment of persistent MV infection, we analysed cells and viruses during the early phase of persistent MV infection in cultured cells. According to previous studies, MV establishes persistent infection in virtually any cell type121314. Thus, we used B95a cells, which are marmoset lymphoblastoid cells, to investigate the mechanism of persistent infection because these cells are highly susceptible to MV infection and can easily achieve 100% infection during persistent infection. In the early phase of MV infection (4 days post-infection [dpi]), most of the cells formed syncytia and died (Fig. 1a). The few remaining cells, however, started to grow again (Fig. 1a). To determine when all of the cells in culture had established a persistent infection, we monitored cellular growth and cell death during the course of MV infection (Fig. 1b and c). Although syncytium formation and cell death faded out at 2 weeks post-infection (Fig. 1a and c), cell growth was severely impaired until 28 dpi when cells started to grow in a manner similar to that of uninfected controls (Fig. 1b). A second crisis occurred between 23 and 28 dpi in all three cultures independently, prior to the re-establishment of cell growth (Fig. 1c). After the establishment of persistent infection, the cell morphology was indistinguishable from that of uninfected cells (Fig. 1a).

View Article: PubMed Central - PubMed

ABSTRACT

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

No MeSH data available.


Related in: MedlinePlus