Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 14;7(1):15588.
doi: 10.1038/s41598-017-15783-x.

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus

Ivana Kutle  1 , Sarah Sengstake  1   2 , Corinna Templin  1 , Mandy Glaß  1   3   4 , Tobias Kubsch  5 , Kirsten A Keyser  1 , Anne Binz  1 , Rudolf Bauerfeind  6 , Beate Sodeik  1 , Luka Čičin-Šain  1   5 , Martina Dezeljin  1 , Martin Messerle  7
Affiliations

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus

Ivana Kutle et al. Sci Rep. .

Abstract

Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cytopathic effect induced by WT MCMV or mutant virus. (a) Monolayers of C127I epithelial cells infected with WT MCMV or the ΔM24-m25.2 mutant were visualized 72 h p.i. by fluorescence and light microscopy. Foci of infected cells were detected utilizing virus-driven GFP expression. (b) The scheme illustrates the genome structure of the mutants. The HindIII cleavage map of the MCMV Smith strain genome is depicted at the top and the genomic region encompassing ORFs M24 to M26 is shown enlarged below. formula image, kanamycin resistance gene; ---, deleted sequences; → , orientation of ORFs; ▪, FRT sites. (c) Cells infected with the indicated viruses were examined by fluorescence microscopy 48 h p.i. Scale bars, 100 µm.
Figure 2
Figure 2
Transcripts and proteins originating from the M25 ORF. (a) Lysates of MEF either mock-infected or infected with the vM25HA virus (at MOI 1) and harvested at the indicated time points were subjected to immunoblotting with an HA antibody. Vimentin served as loading control. Protein size markers (in kDa) are indicated to the left. (b) Total RNA was isolated from NIH 3T3 cells infected with WT MCMV for 6 or 24 h and subjected to Northern hybridization with a 32P-labelled M25-specific probe. (c) The 5′- and 3′-ends of the M25 transcripts were determined by RACE using the RNA samples described in (b) and primers M25-2 and M25-3. Amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Positions of marker bands (b,c) are indicated to the left. (d) Lysates of NIH 3T3 cells prepared 48 h post transfection with plasmids pM25l-HA or pM25s-HA were compared to lysates of NIH3T3 cells infected with vM25HA for 24 h by immunoblotting with an HA-specific antibody. Lysates of mock transfected cells and of cells infected with the ΔM25 mutant for 24 h were included as controls. Vimentin served as loading control. (e) Virions were purified from cultures of cells infected with an MCMV variant expressing an mCherry-tagged small capsid protein or the corresponding ΔM25 mutant and subjected to immunoblot analysis with M25- and mCherry-specific antibodies.
Figure 3
Figure 3
Subcellular localization of M25 proteins during the MCMV infection cycle. (a,b) MEF infected with the vM25HA virus for the indicated time periods were analyzed by confocal microscopy after labeling with antibodies specific for the HA epitope, lamin B and the Golgi protein GM130. Size bar, 10 µm. (c) NIH 3T3 cells were infected with the vM25HA virus. At the indicated time points either total cell lysates (T) or cytoplasmic (C) and nuclear (N) fractions were prepared and analyzed by immunoblotting with the M25-specific antibody. Fractionation was verified for lamin B and β-tubulin.
Figure 4
Figure 4
Virions of the ΔM25 mutant are smaller. Transmission electron microscopy of ultrathin-sections of MEF infected with the ΔM25 mutant (a–f, and h) or WT MCMV (g). Cells infected with the mutant virus (f: overview, N = nucleus, E = extracellular space; (a–e,h): higher magnifications) assemble nuclear capsids (a), primary enveloped capsids (b, arrowheads indicate the outer nuclear membrane), cytosolic capsids in proximity to microtubules (c, arrowheads point at a microtubule), secondary enveloped virus particle (d, arrowheads indicate the vesicle membrane) and extracellular virions (e). Diameters (i) and the sectioned tegumented area (j) of cytoplasmic virions were measured for 10 virions each (g, WT MCMV; h, ΔM25 mutant; arrowheads indicate the viral membrane). Means ± SD are depicted and statistical analysis was done using the paired t-test. ***P < 0.001. Scale bars, 200 nm.
Figure 5
Figure 5
Viral gene expression and genome replication are not affected by the lack of M25 proteins. (a) NIH 3T3 cells were either mock-infected or infected with the indicated viruses (MOI 3.) At the indicated time points lysates were prepared and analyzed by immunoblotting using antibodies specific for the viral immediate-early protein IE1, early protein E1, or glycoprotein B. Vimentin served as loading control. (b) MCMV genome copy numbers present in NIH 3T3 cells (infected as described in (a)) were measured by qPCR at the indicated time points. Graph shows means ± SD of 2 to 3 independent experiments.
Figure 6
Figure 6
Diminished cell-to-cell spread and virus yields of the ∆M25 mutant. (a) Foci formation of infected cells was monitored for 10 days by fluorescence microscopy of MEF monolayers infected with WT MCMV or the ∆M25 mutant at low MOI. Scale bars, 100 µm. (b) Areas of infected cells were measured for WT MCMV (n = 45) and the ∆M25 mutant (n = 45) on day 5 p.i. Infected cells were identified based on virus-driven GFP expression. Bars in the graphs represent medians and statistical analysis was performed by Mann-Whitney two-tailed test. ***P < 0.001. (c) MEF were infected with the indicated viruses at an MOI of 0.1 or 1. Culture supernatants were collected at indicated days after infection and titers quantified by plaque assay. Data points represent means ± SD of triplicate samples. (d) BALB/c mice were infected intraperitoneally with MCMV-GFP-ie1/3 (WT) or MCMV-GFP-ie1/3_ΔM25 (ΔM25), and virus titers in liver, spleen and lungs at day 4 p.i. and in salivary glands at day 21 p.i. were determined by plaque assay. Each dot represents the value for one animal (n = 10 for each group) and bars indicate medians. Statistical significance was calculated by Mann-Whitney two-tailed test. ***P < 0.001.
Figure 7
Figure 7
Morphological changes in WT MCMV and ∆M25 mutant infected cells and in cells transfected with M25 expression plasmids. (a) NIH 3T3 cells were infected with WT MCMV or the ∆M25 mutant or were mock infected. Cells were fixed at the indicated time points, labelled with TRITC-phalloidin and analyzed by confocal microscopy. Positive staining of actin appears black or dark grey. Infected cells are distinguished by virus-driven GFP expression. Size bars, 10 µm. (b) Diameters of the infected cells (treated as indicated in (a)) were determined. (c,d) NIH3T3 cells were either mock transfected or transfected with plasmids pM25l, pM25s and control plasmids encoding the viral proteins M44 or M82 or E.coli ß-galactosidase (lacZ). After 24 h cells were fixed, labeled with Alexa Fluor 488 phalloidin, and antibodies directed against GAPDH and the myc epitope, and examined by confocal microscopy. Transfected cells were identified by the myc signal. Size bars, 10 µm. (c) Cell diameters as determined 24 h after transfection. Bars represent medians. Statistical significance was tested using the Kruskal-Wallis test followed by Dunn’s post hoc test. ***P < 0.001; **P < 0.01. (d) Representative images of cell cultures transfected with M25l, M25s or M44 plasmids. Phalloidin (actin) staining is depicted in the top row and merged signals of phalloidin, GAPDH and myc labeling are shown below. Transfected cells (myc + signal in green) are indicated by arrows.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Condit, R. C. Principles of Virology in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 21–51 (Lippincott Williams & Wilkins, 2013).
    1. Greber UF, Way M. A superhighway to virus infection. Cell. 2006;124:741–754. doi: 10.1016/j.cell.2006.02.018. - DOI - PubMed
    1. Radtke K, Döhner K, Sodeik B. Viral interactions with the cytoskeleton: a hitchhiker’s guide to the cell. Cell. Microbiol. 2006;8:387–400. doi: 10.1111/j.1462-5822.2005.00679.x. - DOI - PubMed
    1. Lyman MG, Enquist LW. Herpesvirus interactions with the host cytoskeleton. J. Virol. 2009;83:2058–2066. doi: 10.1128/JVI.01718-08. - DOI - PMC - PubMed
    1. Taylor MP, Koyuncu OO, Enquist LW. Subversion of the actin cytoskeleton during viral infection. Nat. Rev. Microbiol. 2011;9:427–439. doi: 10.1038/nrmicro2574. - DOI - PMC - PubMed

Substances

LinkOut - more resources