Leu-tRNA, Lys-tRNA and Ser-tRNA deacylation is coupled to translation.

<p>A. Kinetic of [³H]-Leu deacylation on tRNAs extracted from HeLa cells treated or not with sodium arsenite. B. Same as “A” with [³H]-Lys deacylation. C. Same as “A” with [³H]-Ser deacylation.</p

MRS and CRS define two distinct cytosolic compartments.

<p>A. Immunofluorescence staining of HeLa cells. MRS (green) and CRS (red) only partially co-localize. Bar scale: 10 µm. B. Immunoblot of sequential HeLa cells extractions. Cells were first extracted with digitonin (DSC: digitonin soluble compartment), then washed and extracted with NP-40 (DRC: digitonin resistant compartment). Membranes were blotted for MRS, CRS and ribosomal P proteins. C. HeLa cells were pre-treated for 2 h with MG132 or for 30 min with either arsenite or pactamycin. Then cells were labeled with [³⁵S]-Cys or [³⁵S]-Met. Half of each sample was used to extract total proteins and cpm were counted. 100% cpm was determined using untreated HeLa cells. For [³⁵S]-Met: 100% = 982785 cpm. For [³⁵S]-Cys: 100% = 371828 cpm. D. The other half of each sample from “C” was used. Total RNAs were extracted and cpm counted. 100% cpm was determined using untreated HeLa cells. For [³⁵S]-Met: 100% = 288248 cpm. For [³⁵S]-Cys: 100% = 69183 cpm. E. The same samples used in “D” were loaded on acid gels to visualize either [³⁵S]-Met-tRNAs or [³⁵S]-Cys-tRNAs. The two bands for [³⁵S]-Met-tRNAs correspond to the mitochondrial tRNA^Met and the nuclear encoded tRNA^Met.</p

Cys-tRNA deacylation is uncoupled from translation.

<p>A. Kinetics of [³⁵S]-Met charging on tRNAs with or without pre-treating HeLa cells with sodium arsenite. The same samples were both analyzed by beta counter and loaded on a native acid gel. B. Same as “A” with [³⁵S]-Cys charging. C. Kinetics of [³⁵S]-Met deacylation on tRNAs extracted from HeLa cells pretreated or not with sodium arsenite. This panel shows two different graph representations: “raw” data using Cpm/µg of total RNA and representing one experiment or calibrated data defining time 0 as 100% of [³⁵S]-Met charged tRNAs and averaging 2 distinct experiments. The same samples were both analyzed by beta counter and loaded on native acid gels. D. Same as “A” with [³⁵S]-Cys deacylation.</p

Compensatory NA mutations decrease NA activity.

<p>Three-dimensional view of HA trimer (a) and NA tetramer (b). Mutations found in individual viruses are in different colors (yellow = CV1, orange = KV2, green = JV9). Sialic acid (a) and the NA active site (b) are shown in red. (c) NA activities of viruses +/− Triton-X were determined using a colorimetric assay. The amount of glycoproteins incorporated in purified virions was determined by ELISA (d), western blots (e), and total protein blots (f). *, p<0.05 compared to PR8.</p

Compensatory mutations alter NA drug sensitivity and antigenicity.

<p>Viruses were pre-incubated with zanamivir (a) or oseltamivir (b) and NA activity was determined. (c) Relative binding affinities of 3 control Abs (NA polyclonal Ab, HA mAb, and NP mAb) and 4 anti-NA mAbs were determined using an ELISA assay. Ab titrations were performed and relative K_d values were transformed into relative colors using the matrix2png software. Black indicates high binding affinity and white indicates low binding affinity.</p

Enterovirus D-68 Infection, Prophylaxis, and Vaccination in a Novel Permissive Animal Model, the Cotton Rat (<i>Sigmodon hispidus</i>)

<div><p>In recent years, there has been a significant increase in detection of Enterovirus D-68 (EV-D68) among patients with severe respiratory infections worldwide. EV-D68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for EV-D68, a comprehensive understanding of the pathogenesis and immune response against EV-D68 has been hampered. Recently, it was shown that EV-D68 has a strong affinity for α2,6-linked sialic acids (SAs) and we have shown previously that α2,6-linked SAs are abundantly present in the respiratory tract of cotton rats (<i>Sigmodon hispidus</i>). Thus, we hypothesized that cotton rats could be a potential model for EV-D68 infection. Here, we evaluated the ability of two recently isolated EV-D68 strains (VANBT/1 and MO/14/49), along with the historical prototype Fermon strain (ATCC), to infect cotton rats. We found that cotton rats are permissive to EV-D68 infection without virus adaptation. The different strains of EV-D68 showed variable infection profiles and the ability to produce neutralizing antibody (NA) upon intranasal infection or intramuscular immunization. Infection with the VANBT/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. Intramuscular immunization with live VANBT/1 or MO/14/49 induced strong homologous antibody responses, but a moderate heterologous NA response. We showed that passive prophylactic administration of serum with high content of NA against VANBT/1 resulted in an efficient antiviral therapy. VANBT/1-immunized animals showed complete protection from VANBT/1 challenge, but induced strong pulmonary Th1 and Th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. In conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.</p></div

Intramuscular immunization with VANBT induces both Th1 and Th2 cytokines upon virus challenge.

<p>Female cotton rats were mock-immunized i.m. on day 0 and at 3 weeks, infected with VANBT on day 0, or immunized i.m. on day 0 and at 3 weeks with UV-VANBT, or VANBT, using 10⁶ TCID₅₀/100 μl of virus. At 7 weeks, all animals were challenged i.n. with 10⁶ TCID₅₀ of VANBT and sacrificed at 10 h or 48 h p.i. Relative mRNA expression profiles of IFN-β (A), Mx-2 (B), IP-10 (C), IFN-γ (D), IL-6 (E), IL-4 (F), IL-5 (G), and IL-13 (H) at either 10 or 48 h p.i. in the lung tissues were measured by qRT-PCR. Results were calculated as fold-induction over uninfected (naïve) animals and expressed as geometric means ± SE. Results are representative of two independent experiments, n = 5. * p<0.05 for each group compared with mock-immunized group, #p<0.05 when VANBT/i.m. compared to VANBT/i.n. and ⦁p<0.05 when VANBT/i.m. compared with UV-VANBT/i.m.</p

Intramuscular immunization with EV-D68 protects against VANBT challenge.

<p>(A) Schematic representation of infection and immunization regimen. Female cotton rats were mock-immunized i.m. on day 0 and at 3 weeks, infected with VANBT on day 0, or immunized i.m. on day 0 and at 3 weeks with UV-VANBT, ATCC, VANBT, or MO/49 using 10⁶ TCID₅₀/100 μl of virus. At 7 weeks, all animals were challenged i.n. with 10⁶ TCID₅₀ of VANBT and sacrificed at 10 h or 48 h p.i. (B) Viral titers in the nose and lung of animals sacrificed at 10 h p.i. n = 5 per group. Data are representative of two independent experiments. * <i>p</i><0.05 where each group is compared with mock-immunized group.</p

Intramuscular immunization with live EV-D68 strains induces virus-specific homologous NA titers.

<p>(A) Female cotton rats were immunized i.m. with 10⁶ TCID₅₀/100 μl of indicated virus on day 0 and boosted 3 weeks after the first immunization. Serum samples were obtained at 3 week (before boosting) and 7 weeks after the first immunization and homologous serum NA titers were determined. The sera were assayed in duplicate and NA titer is expressed as Log₂ geometric mean ± SE. n = 10–15 from two independent experiments, ***p< 0.001 for 3 weeks NA titer compared with 7 weeks NA. (B) Passive transfer of VANBT immune sera protects animals from VANBT challenge. Animals were treated intraperitoneally with 0.5 ml 1 x PBS (mock) or serum from animals immunized i.m. with either UV-VANBT, VANBT, or MO/49. The following day, animals were challenged i.n. with 10⁶ TCID₅₀ of VANBT and euthanized 10 h later to determine nose and lung viral titers. n = 4–5 per group. * p<0.05 for each group compared with mock group.</p

Homologous and Heterologous serum NA titer in EV-D68 immunized cotton rats.

<p>Homologous and Heterologous serum NA titer in EV-D68 immunized cotton rats.</p