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Results: 5

1.
Figure 2

Figure 2. Acute defect in virus production is restored by viral cyclins.. From: Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins.

(A) Schematic representation of infections for lung titers and pathology in IFN-g-/-mice. Five mice were included in each group. (B) Histological analyses of lung tissues from mock, gHV-cycKO, gHV-cycV, gHV-cycK, gHV-cycA, gHV-cycE, gHV-cycD3 and gHV-cycD2-infected mice. Representative H&E images show clear and healthy airspaces in mock infected image and examples of neutrophil infiltrates indicated by arrowheads. Insets and upper left panel magnification ×40, all other panels magnification ×10. (C) Viral titers in the lungs were determined by plaque assay. Limit of detection is indicated by dotted line. Titers were statistically significant (*p<0.05) when compared to gHV-cycKO, as determined by a one-way ANOVA test.

Katherine S. Lee, et al. PLoS Pathog. 2012 February;8(2):e1002496.
2.
Figure 3

Figure 3. Both viral and mammalian cyclin-expressing viruses confer endothelial cell survival and persistence.. From: Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins.

(A) Schematic representation of persistent infection of endothelial cells for viability determination. (B) MB114 endothelial cells were infected with recombinant viruses for 6 days, at which time non-adherent cells were collected and analyzed for viability by dual PI and annexin V staining. Flow cytometry zebra plots are representative of 3 independent experiments from gHV-cycV, gHV-cycK, gHV-cycA, gHV-cycE, gHV-cycD3 and gHV-cycD2 virus infections. (C) Bar graph of mean viability (annexin V-/PI-) from flow cytometric analysis of 3 independent experiments. Data was found to be statistically significant between gHV-cycKO compared to WT (*p = 0.001), gHV-cycV (*p = 0.004), gHV-cycK (*p = 0.001), gHV-cycE (*p = 0.001) and gHV-cycA (*p = 0.001) as determined by unpaired t-test.

Katherine S. Lee, et al. PLoS Pathog. 2012 February;8(2):e1002496.
3.
Figure 4

Figure 4. Viral cyclins and cyclin D3 compensate in reactivation from latency.. From: Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins.

(A) Schematic representation of infections and determination of ex vivo reactivation in C57BL/6 mice. (B–D) Limiting-dilution ex vivo reactivation from latency of PECs from mice infected with (B) compiled WT, gHV-cycKO and recombinant cyclin viruses, (C) gHV-cycV, gHV-cycK and gHV-cycD3 viruses that complement in reactivation and (D) gHV-cycA, gHV-cycE and gHV-cycD2 viruses that do not complement in reactivation. Data represent the mean ± SEM of the indicated number of independent experiments, with each experiment containing cells pooled from three to five mice and each experiment including the gHV-cycKO virus. The dashed line is at 63%, the value which was used to calculate the frequency of reactivating cells as indicated by a Poisson distribution. Reactivation frequencies were as follows: WT-gHV 1/8729, gHV-cycV 1/13152, gHV-cycK 1/19952, gHV-cycD3 1/25118; and less than 1/80000 for gHV-cycKO, gHV-cyc, gHV-cycE and gHV-cycA. Frequencies of reactivation were significantly increased by gHV-cycV (*p = 0.01), gHV-cycK (*p = 0.02) and gHV-cycD3 (*p = 0.03) compared to gHV-cycKO, as determined by paired t-test.

Katherine S. Lee, et al. PLoS Pathog. 2012 February;8(2):e1002496.
4.
Figure 5

Figure 5. Proposed model for viral cyclin requirements in virus infection.. From: Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins.

Recombinant knock-in viruses with various viral or mammalian cyclins expressed from the endogenous gHV68 viral cyclin locus are depicted at upper left. Genetic complementation analysis of this panel of viruses demonstrated that the viral cyclins of gHV68 and of KSHV are functionally conserved and are unique in their ability to support all cyclin-dependent aspects of infection, depicted as three different panels. Further, mammalian cyclins able to function in virus infection comprised two genetically distinct groups, based on their ability to complement either persistent infection in endothelial cells (lower left) or reactivation from latency in lymphocytes (lower right). Finally, we propose that the unique capacity of the viral cyclins to support acute virus production and lethal pneumonia in the lungs of immune deficient mice does not reflect another distinct genetic requirement of viral cyclins. Rather, we hypothesize that both persistent infection and reactivation from latency contribute to optimal acute virus production in the lungs (upper right), as indicated by the combination of blue and yellow.

Katherine S. Lee, et al. PLoS Pathog. 2012 February;8(2):e1002496.
5.
Figure 1

Figure 1. A panel of recombinant viruses express various cyclins under control of a uniform expression cassette.. From: Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins.

(A) Schematic representation of the recombinant viruses generated using BAC recombination. Genome coordinates of the v-cyclin ORF (bp 103181 to 102426) are based on the gHV68 WUMS sequence. The diamond denotes the location of the single 34 bp loxP site. The N-terminal 3x-FLAG epitope tag is shown as a blue rectangle. The cyclin region probe spans bp 101654 to 105377, and the left end probe spans bp 11100 to 14026. At the left end of the viral genome are the locations of the NotI and NsiI sites used to diagnose possible deletions in this region of the genome. (B) Quantitative RT-PCR of RNA isolated from 3T12s infected for 12 (n = 2) and 48 (n = 3) hours with WT and recombinant cyclin viruses. Values are mean ± SD. One way ANOVA Bonferroni's Multiple Comparison Test determined no statistical differences between the samples. (C) Recombinant cyclin viruses express specific cyclin proteins. Lysates from 3T12s infected with two independent clones of recombinant cyclin viruses (designated at the right) for 24 hours were transferred and probed with the antibodies indicated above blots. The same number of cell equivalents was used and the data is representative of multiple experiments. (D) Recombinant cyclin viruses express each cyclin protein abundantly during lytic infection of 3T12 fibroblasts. Lysates from 3T12s infected for 24 hours were transferred and probed with antibodies to beta-actin, followed by the Flag epitope. 10 µg of lysate was loaded per lane and data is representative of multiple experiments. (E) Multistep replication of recombinant cyclin viruses. 3T12 cells were infected with recombinant cyclin viruses expressing viral or mammalian cyclins at a MOI of 0.05 PFU/cell and harvested at 0, 12, 24, 48, 72, 96 and 120 hours post-infection. Both cells and supernatants were analyzed by plaque assay. Data shown are from two independent infections, from which at least three plaque assays were performed per virus, with each sample being measured in triplicate. Mean ± SEM are shown. Dotted line indicates the limit of detection of the assay (50 PFU).

Katherine S. Lee, et al. PLoS Pathog. 2012 February;8(2):e1002496.

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