Phosphorylation of Single Stranded RNA Virus Proteins and Potential for Novel Therapeutic Strategies

Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections

The role of IKKβ in Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKβ component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKβ component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKβ in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKβ function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKβ function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKβ for VEEV replication, we over-expressed IKKβ in cells and observed an increase in viral titers. In contrast, studies carried out using IKKβ(-/-) cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKβ may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKβ protein may be critically involved in VEEV replication

Bortezomib decreases intracellular viral RNA.

<p>A) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were pre-treated with 0.1μM Bortezomib or DMSO for 2 hours and then infected with TC-83 (MOI: 0.1) for 1 hour. At 0, 2, 4, 6, and 8 hours post infection cells were lysed with MagMAX-96 Total RNA Isolation Kit. As controls, cells were either pre-treated with 0.1μM Bortezomib or DMSO for 2 hours, then infected with TC-83 (MOI: 0.1) and the cells lysed at 24 hours post infection. Levels of viral RNA were quantified by q-RT-PCR using VEEV specific primers. The cycling conditions and probe primer pair sequences are described in the methods section. The graphs represent an average of 2 independent experiments where each experiment was performed in triplicate. Standard deviations were calculated accordingly. p≤0.0001 (**). U87MG cells were seeded onto #1.0 coverslips in 24-well plates at a density of 54,000 cells per well. Cells were pretreated with Bortezomib for 2 hours and then infected with TC-83 (MOI: 5) (B and C). B) After 8 hours of infection, the cells were fixed and processed for RNA FISH as described in the materials and methods section. Following FISH staining, cells were imaged using a Zeiss 700 confocal microscope with a 20X objective and at least 10 high-powered fields (HPFs) were obtained for each sample. The percentage of infected to uninfected cells was calculated per HPF for each condition. A representative 20X objective image is shown with viral RNA in red and nuclei in blue. C) After 0, 4 and 8 hours of infection, the cells were fixed and processed for RNA FISH as described in the materials and methods section. Following FISH staining, cells were imaged using a Zeiss 700 confocal microscope. A representative 63X objective image is shown with viral RNA in red and nuclei in blue. D) VERO cells were infected with TC-83 or Rift Valley Fever Virus (RVFV) (strain MP-12) for 24 hours. Cells were then processed for RNA FISH and stained with VEEV vRNA specific FISH probes as described in the materials and methods section. Following the RNA FISH staining, the VEEV infected cells were immunostained with an anti- VEEV capsid antibody and the RVFV infected cells were immunostained with an anti-RVFV nucleocapsid antibody. Nuclei were detected using DAPI. Images are shown as condensed Z-stacks. The images are representative of 2 independent experiments performed in duplicate.</p

VEEV capsid protein is ubiquitinated on K48.

<p>A) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 1, 2, 5 and 6 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 replicates in an experiment. Red boxes are parts of the image that have been zoomed in and displayed in Z-stacks. B) Number of infected U87MG cells showing co-localization tabulated. C) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were untreated (Mock), DMSO treated or Bortezomib treated (0.1μM) for 2 hours. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 2 hours post infection, cells were fixed and processed as described in the materials and methods section. The cells were probed with K48 ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 independent experiments performed in duplicate. D) Number of infected U87MG cells showing co-localization tabulated. E) U87MG cells were treated with Bortezomib (0.1μM) or DMSO for 2 hours and then infected with TC-83 (MOI: 5) for 1 hour. At 2 hours post infection cell lysates were collected, lysed and quantified. Equal amounts of total protein were immunoprecipitated with capsid antibody and resolved by SDS-PAGE and subsequently immunoblotted for K48 ubiquitin (top panel) and capsid (bottom panel). The image is representative of 2 independent experiments. Protein bands were quantified using Image J software and normalized to capsid. Average percent differences of 2 independent experiments are depicted graphically.</p

Other proteasomal inhibitors decrease TC-83 multiplication in U87MG cells.

<p>A) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were pre-treated with MG132 at 0.01μM, 0.1μM, or 1μM concentrations for 2 hours. Treated cells were infected with TC-83 at MOI: 0.1 for 1 hour. Supernatants were collected 24 hours post infection and infectious viral titers (PFU/mL) were assessed by plaque assay. B) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were treated with either DMSO or varying concentrations of MG132. Cell viability was determined 24 hours post-treatment using Cell-Titer-Glo Reagent as per manufacturer’s instructions. The graphs are representative of 3 independent experiments, each performed in triplicate. Standard deviations were calculated from 3 independent experiments and are represented thusly. C) U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were infected in triplicate with TC-83 at MOI: 0.1 for 1 hour. Infected cells were treated with 1μM of MG132 at 2 hour intervals from 0–8 hours post infection. As controls infected cells were treated with DMSO. Supernatants were collected 24 hours post infection and analyzed by plaque assay for infectious viral particles (PFU/mL). The arithmetic means are illustrated graphically. The graph is representative of 2 independent experiments performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*).</p

Inhibition of wild type alphaviruses by Bortezomib.

<p>U87MG cells were pre-treated with Bortezomib at 0.1μM for 2 hours and then infected with VEEV TrD, EEEV strain GA97, and WEEV California 1930 strain at an MOI: 0.1 for 1 hour. Supernatants were collected 24 hours post infection and analyzed by plaque assay for infectious viral particles (PFU/mL). The arithmetic means are illustrated graphically. The graph is representative of 3 independent experiments where each experiment was performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*).</p

Bortezomib inhibition is independent of viral load.

<p>U87MG cells were seeded in a 96-well plate at a density of 10,000 cells per well. Cells were pre-treated with Bortezomib (0.1μM) or DMSO in triplicate for 2 hours. Treated cells were infected with TC-83 at MOI: 0.1 (A) MOI: 1 (B) or MOI: 5 (C). The viral inoculum was removed and replaced with conditioned media. At 6, 12, 18 and 24 hours post infection supernatants were collected and analyzed by plaque assay. The graph is representative of 2 independent experiments performed in triplicate. Standard deviations were calculated accordingly. p<0.05(*). p≤0.0001 (**). U87MG cells were seeded in a 12-well plate at a density of 100,000 cells per well. Cells were pre-treated with Bortezomib (0.1μM) or DMSO for 2 hours. Treated cells were infected with TC-83 at MOIs (0.1, 1 or 5) (right panels of A, B and C respectively). The viral inoculum was removed and replaced with conditioned media. At 6, 12, 18 and 24 hours post infection cell lysates were collected and analyzed by western blot. The images are representative of 2 independent experiments.</p

Ubiquitinated capsid in virions.

<p>VEEV-containing supernatants obtained from infected VERO cells were subjected to sucrose density centrifugation as described in the materials and methods section. A) The collected fractions were analyzed by plaque assay and are represented graphically. Fraction 3 with the highest titer of virus was used for immunoprecipitation with capsid antibody and an isotype IgG as a control. Immunoprecipitated samples were resolved by SDS-PAGE and subsequently immunoblotted for ubiquitin (top panel) and capsid (bottom panel). The image is representative of duplicate immunoprecipitation runs.</p

Ubiquitination of a viral protein.

<p>A) U87MG cells were transfected with HA-Ub for 24 hours and then infected with TC-83 (MOI: 5) for 1 hour. At 6 hours post infection, cell lysates were collected and quantified for protein concentration. Two milligrams of total protein was immunoprecipitated with HA probe antibody and mouse IgG as a control. LC/MS-MS was performed and the results tabulated. B) U87MG cells were seeded in an 8-well chambered slide at 20,000 cells per well. The cells were uninfected (Mock) or infected with TC-83 at an MOI of 10. At 2 hours post transfection, cells were fixed and processed as described in the materials and methods section. The cells were probed with ubiquitin and capsid antibodies followed by incubation with Alexa-Fluor 488 and Alexa-Fluor 568 respectively. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U with a 60X objective and are representative of 2 independent experiments performed in duplicate. C) U87MG cells were infected with TC-83 (MOI: 5) for 1 hour. At 6 hours post infection, cell lysates were collected and quantified for protein concentration. Two milligrams of total protein was immunoprecipitated with ubiquitin antibody and an isotype IgG as a control. Immunoprecipitated samples were resolved by SDS-PAGE and subsequently immunoblotted for capsid (top panel) and ubiquitin (bottom panel). The image is representative of 3 independent experiments.</p