Selective incorporation of pERK2 in RRV virions.
In addition to detecting over 30 virally encoded proteins comprising RRV particles (51
), analysis of our more recent tandem mass spectrometry (MS/MS) data using highly purified particles also revealed the presence of a number of cellular proteins (see Table S1 in the supplemental material), including a mitogen-activated protein kinase (MAPK), ERK2 (). On each of two separate viral preparations, MS/MS analyses revealed 9 and 12 distinct tryptic peptides, respectively (for a total of 42.5% coverage), mapping to ERK2 (, shaded in gray). Although previous proteomic analyses of other herpesviruses have not reported the presence of such signaling molecules within mature gammaherpesvirus virions (5
), we reasoned that RRV-associated ERK2 might simply arise from elevated intracellular concentrations of this signaling molecule during RRV assembly. Of note, however, this approach failed to detect evidence of ERK1 within the virions.
Fig 1 Preferential incorporation of ERK2 into RRV particles. (A) MS and MS/MS analyses of sucrose gradient-purified RRV particles identified multiple tryptic peptides (shaded) that mapped to ERK2. MS/MS analysis of these samples following titanium enrichment (more ...)
To confirm the MS data, we separated the virion-associated proteins of gradient-purified RRV by SDS-PAGE and performed immunoblot analyses, using a quantitative, nonenzymatic infrared detection system (see Materials and Methods) to probe for three known virally encoded structural proteins, the major capsid protein (MCP/ORF25), a tegument protein encoded by ORF45, and the small capsomer-interacting protein (SCIP/ORF65), as well as for ERK1 and ERK2 (ERK1/2), using an antibody that recognizes both isoforms equally (, lane 1). The results of this representative experiment (performed using >5 separate viral preparations) indicated that all three structural proteins were evident in the particle, along with a relative abundance of the faster migrating of the two ERK species. The immunoblot results demonstrated that the virions contained only a trace amount of the slower-migrating species. Due to the migration of these two ERK bands, we assumed that the top band represented ERK1 (p44) and the dominant bottom band represented ERK2 (p42). To confirm their identities, we stripped the blot and reprobed with a monospecific antibody to ERK2 (). The bands of ERK2 were superimposable on the lower bands of each ERK pair in . The abundance of ERK2 compared to ERK1 within the virions provided a possible explanation for our consistent detection of only the former isoform in multiple MS analyses of the virions.
To determine if the ERK2 bias within virions resulted from preferential incorporation of one isoform over the other or, instead, simply reflected their relative abundances within the cell, we also analyzed, in parallel, the infected and uninfected cell lysates (, lanes 2 and 3, respectively), probing the blots for the two ERK isoforms along with MCP, ORF45, and SCIP. ERK1, ERK2, and Ran (the cellular loading control) were all present in both the infected and uninfected cell lysates, while the viral proteins were evident only in infected cells. We noted that uninfected cells had approximately equal levels of ERK2 and ERK1 (, lane 3), while infected cells had slightly more ERK2 than ERK1 (, lane 2), but, as we noted above, purified virions showed clear evidence of ERK2, with only trace amounts of ERK1 (, lane 1). This suggested that the virion content of the two ERK isoforms was not simply a sampling of the intracellular environment of the producer cell.
We also noted an upward mobility shift in the ERK2 band present in the infected compared to uninfected cells that was also evident and even further shifted in the virions ( and ). Since a variety of viruses, including KSHV, induce ERK phosphorylation upon infection (3
), we hypothesized that this mobility shift in the ERK2 protein band might similarly be due to its phosphorylation status. To begin to test this idea, we stripped the ERK blot and reprobed with an antibody that specifically recognizes dually phosphorylated (fully activated) ERK (pERK in ). In the absence of activating stimuli, uninfected cells contained only minimal levels of pERK, but infected cells, and purified virions released from these cells, showed markedly increased levels of pERK2 and a relative paucity of pERK1 (compare lanes 1 to 3 in ). Of note, persistent intracellular activation of ERK required infectious virions. In contrast, exposure to UV-inactivated RRV led to a rapid but transient rise in the level of intracellular pERK2 that returned to baseline by 2 to 4 h (E. N. Woodson and D. H. Kedes, unpublished data). After normalizing for gel loading differences, we found that the levels of pERK1 in infected cells were only minimally higher than in uninfected controls (compare upper pERK bands and their respective Ran bands in lanes 2 and 3 of to panel B data). Since the pERK2 bands were superimposable on the shifted ERK2 bands (lanes 1 and 2 in and ), we concluded that the lower ERK bands in virions and infected RhF lysates contained pERK2 with or without additional posttranslational modifications.
To determine if ERK2 was phosphorylated at any other amino acids in addition to those recognized by the pERK antibody (pT185 and pY187), we used MS/MS to analyze highly purified virions after first enriching for phosphopeptides (see Materials and Methods). The majority of the phosphopeptides mapped to virally encoded structural proteins, but, notably, the only sites mapping to ERK2 were the MEK phosphorylation sites on ERK2 (T185 and Y187) (63
) ( and highlighted residues within the annotated sequence in panel 1A) whereas no phosphopeptide(s) mapped to ERK1. In contrast, even though phosphatase treatment eliminated the ability of the pERK-specific antibody to recognize the ERK2 species in the immunoblots, reprobing with ERK1/2 antibody showed that this treatment failed to reverse the shifts (not shown), suggesting that ERK2 species within the infected RhF and virions also underwent one or more additional posttranslational modifications that we have, to date, been unable to identify. Nevertheless, together, these data indicated that RRV infection led to activation of ERK2 and the preferential incorporation of this isoform during viral assembly. Finally, to further address whether virion incorporation of pERK2 was directed rather than reflecting a random incorporation of any one of the many activated signaling molecules within infected cells, we probed for another phosphoprotein, pp38. This activated protein also localizes to the nucleus in response to a variety of stimuli (6
) and became phosphorylated following RRV infection (, lane 2). However, in contrast to our findings with pERK2, we were unable to detect pp38 within the virion (, lane 1). Of note, we were similarly unable to detect Jun N-terminal protein kinase (JNK) within the virion (not shown). Taken together, these data argue for the specificity of pERK2 packaging within mature virions.
pERK2 localizes to the tegument of RRV.
Since cellular proteins within herpesviruses tend to localize to the tegument layer of the particle (5
), we hypothesized that pERK2 would reside within this same layer in RRV. To determine the specific location of pERK2, we subjected virions to different combinations of proteolytic as well as detergent treatments, followed by gradient purification of the resulting particles and analysis by immunoblotting. This approach served to localize pERK2 within the virion while also ensuring that it was not simply adhering nonspecifically to the virion surface. Since each viral capsid contains 955 molecules of MCP (20
), we probed for and then quantified MCP in each sample to normalize for particle number in parallel to determining the relative abundance of pERK2 under each condition (). Again, untreated RRV particles predominantly contained pERK2 (, lane 1), and this signal persisted even after treatment with proteinase K (PK) (, lane 2), which removes proteins that adhere nonspecifically to the virion surface (51
). As a control, we treated lysates from RRV-infected cells (48 h p.i.; MOI of 10) with increasing concentrations of PK under identical treatment conditions, and intracellular pERK was degraded (not shown). This suggested that pERK was protected from PK by the viral envelope. Next, we used Triton X-100 (, lane 3), a nonionic detergent, to solubilize the viral envelope and found that pERK2 remained associated with the purified particle. Even at higher-temperature incubations with Triton X-100 (1 h at 37°C), over 50% of the pERK2 signal remained with the purified particle (not shown). Together, these findings suggested that pERK2 was present within the tegument or, potentially, within the capsid. To distinguish between these two remaining possibilities, we derived untegumented capsids directly from the nuclei of infected cells. Since tegumentation occurs after egress from the nucleus, we reasoned that if pERK2 were in the tegument, the nuclear-derived particles would lack pERK2. We found this to be the case (, lane 4). Another known tegument protein encoded by ORF52 was also absent in the nuclear capsids, as we had anticipated from our earlier work (reference 51
and data not shown).
ERK activation is important in lytic viral gene expression.
Though this paper is the first to report the incorporation of ERK in a herpesvirus particle, previous studies have shown that activation of the MEK/ERK pathway is critical for the production of other herpesviruses (56
). Blocking ERK activation with either pharmacological or molecular inhibition of MEK (e.g., siRNA or dominant-negative constructs) (24
) completely abrogated virus production. Since our data demonstrated that RRV infection activated ERK (), we hypothesized that activation of this pathway was also essential for RRV production. To test this, we used the MEK inhibitor U0126 (23
) to block ERK activation prior to and during RRV infection and then, with an immunoblot of cell extracts, measured the expression of viral proteins MCP, ORF45, and SCIP as well as cellular proteins ERK1/2, pERK1/2, and Ran. To ensure that U0126 retained its MEK inhibition for the duration of the experiment, we transiently stimulated RhF with the phorbol ester TPA 48 h after treating the cells with either the drug vehicle (DMSO) or U0126 (, lanes 1 and 2). ERK activation (pERK1/2) was evident in cells treated with TPA in the absence of drug but greatly diminished in cells in the presence of drug, demonstrating its efficacy throughout the duration of the experiments. Even when overloaded on the immunoblot, lysates from uninfected cells showed only trace amounts of ERK activation (, lane 3). In lysates from infected cells pretreated with vehicle, all three viral proteins were present and ERK was expressed and activated (pERK) (, lane 4). However, in lysates from RRV-infected cells in the presence of U0126, little to no ERK activation was evident and these cells showed only a trace amount of MCP and no evidence of the other viral proteins on the immunoblot (, lane 5). U0126 had no significant effect on cell viability even after 48 h of treatment (98% viability with DMSO alone compared to ~93% viability with the drug). Moreover, U0124, a structurally related but inactive analog of U0126, did not inhibit ERK activation and did not affect expression of the viral lytic proteins and viral titers compared to the results seen with infected cells treated with DMSO alone (data not shown).
Fig 3 The MEK inhibitor U0126 blocks the activation of ERK in RRV infection, inhibiting viral protein expression and virus production. (A) RhF were stimulated with TPA after 48 h of culture in the presence of drug vehicle DMSO (lane 1) or the MEK inhibitor (more ...)
To further evaluate the effects of blocking ERK activation on RRV production, we also monitored the cells by phase microscopy throughout infection. By 48 h, the uninfected, untreated monolayers remained intact, displaying swirls of elongated cells, characteristic of quiescent fibroblasts in culture (, panel i). In contrast, infected cells treated with vehicle showed typical signs of lytic infection, with the majority of the monolayer disrupted with rounded cells (, panel ii). In infected cells treated with drug, however (, panel iii), the monolayer was mainly left intact, much like uninfected cells, with only a few foci of rounded cells, suggesting that the drug was blocking nearly all virus production. To quantify more precisely the effect this block of ERK activation had on viral production, we determined the titer of the virus released under each condition (see Materials and Methods) (). In three separate experiments, absolute titers differed but, invariably, those of the U0126-treated RRV-infected cells were more than 2 logs lower than those from infected cells treated with vehicle alone (set to a relative value of 100). We also used qPCR to determine viral genome copy numbers in the supernatants of infected cells treated with or without U0126. As we expected based on the titer data (), the viral genome copy number was significantly lower (~30-fold; P = 0.0001) in cells treated with U0126 compared to those treated with the DMSO control (). Collectively, these data pointed to a critical role for ERK activation in the viral life cycle of RRV, though they did not distinguish at which stage this activation was most important.
Sharma-Walia et al. have shown that ERK is activated at various time points after KSHV infection, including glycoprotein engagement of cellular receptors and entry; however, this activation was most necessary for the establishment of infection and early viral gene transcription (69
). To determine if this is also a requirement for RRV, we monitored the effects of U0126 on viral entry by measuring intranuclear viral DNA postinfection in the presence of the drug. Using qPCR, we found no significant difference in the numbers of intranuclear viral genomes 4 h postinfection, whether in the presence or absence of U0126 (; P
= 0.07). In contrast, U0126 dramatically decreased the level of the immediate early gene ORF50 (). Not surprisingly, we also found that early (ORF37) and late (ORF25) lytic mRNA levels were also greatly decreased in U0126-treated cells (). As an additional control, we treated infected cells simultaneously with U0126 and a viral DNA polymerase inhibitor, phosphonoacetic acid (PAA), which blocks viral DNA replication, thereby preventing viral genome amplification. The decrease in levels of all lytic transcripts was even greater in the presence of PAA, though these differences did not reach statistical significance (). Together, these data indicated that RRV, like KSHV, depends on the activation of ERK activation for immediate early gene expression.
Fig 4 Expression of lytic RRV genes but not episome delivery to the nucleus is dependent on ERK activation. (A) RhF were infected in the absence or presence of U0126. At 4 h p.i, nuclei were isolated and DNA was extracted and analyzed by qPCR using RRV ORF45 (more ...) Altering the absolute or relative levels of intracellular ERK isoforms produces minimal changes in intracellular viral protein production.
Although initial studies often assumed that ERK1 and ERK2 were interchangeable and served redundant functions (68
), more recent data suggest that they may have distinct roles (39
). Since we noted a marked and reproducible bias toward ERK2 activation in RRV infection (, lane 2, and , lane 4), we chose to test whether ERK2 plays a critical and nonredundant role in RRV infection and virion production. We used a nontargeting siRNA as a control (siCNL) to establish the levels of ERK1 and ERK2 during RRV infection, pretreating and leaving the siRNA in the media during the 48-h infection, and then collected the whole-cell lysate (, lane 1). In nearly all experiments, the steady-state levels of ERK2 were slightly higher than those of ERK1 in infected cells treated with or without siRNA. To establish relative ERK1 and ERK2 levels among repeated experiments with RRV-infected RhF, we chose to set the ERK2 level from siCNL-treated cells to 1.0 and compared all other ERK1 and ERK2 values to it. (Note that the anti-ERK antibody equally detects both isoforms.) Pretreatment of the cells with siRNA directed to ERK1 (siERK1; , lane 2) led to a marked (~84%) knockdown in ERK1 without a significant change in ERK2. siRNA knockdown of ERK2 (siERK2; , lane 3) resulted in a similar (~86%) decrease in ERK2 and a slight (~19%) but not statistically significant increase in ERK1. Finally, siRNA targeting of both ERK1 and ERK2 resulted in ~87% and ~83% knockdown of ERK1 and ERK2, respectively (, lane 4). shows the graphical representation of these data from four independent experiments. Since the loss of one isoform did not result in a compensatory increase in the expression of the other, the results were consistent with the possibility that ERK1 and ERK2 have functionally distinct roles.
Fig 5 Differential suppression of ERK1 and ERK2 isoforms only minimally affects intracellular viral protein production. (A) RhF were transfected with nontargeting siRNA (siCNL), siERK1, siERK2, or siERK1 plus siERK2, as indicated. At 24 h posttransfection, (more ...)
In light of previous studies performed with KSHV that showed that several ERK substrates bind to viral promoters to modulate viral protein expression (56
), we predicted that knockdown of one or both of the ERK isoforms would downregulate the intracellular levels of structural viral proteins. However, we found that targeting of ERK1, ERK2, or both by siRNA knockdown failed to profoundly downregulate the expression of these structural viral proteins compared to the siRNA control-treated cell results (, lanes 2 to 4 versus lane 1), with the exception of a modest decrease in levels of ORF45 in the dual ERK1 and ERK2 knockdown (, lane 4). depicts quantitatively the combined data from four experiments, all normalized to Ran. Thus, although we found ERK activation to be essential for lytic viral gene and, thus, protein expression ( and ), the siRNA ERK knockdown results decribed above demonstrated that relatively large changes in total ERK levels had only minor effects on the steady-state levels of viral structural proteins within infected cells, at least at 48 h postinfection.
Residual pools of pERK2 remain following knockdown of ERK1 and ERK2 during RRV infection.
Initially, the MEK inhibitor and siERK results seemed discordant. We were surprised to find persistent production of the viral structural proteins in siERK-treated cells, since blockade of ERK activation essentially abrogated viral protein and virion production (). Though each siERK condition led to marked knockdowns in intracellular ERK during RRV infection, we hypothesized that this might have proportionally lower effects on intracellular pools of pERK. If so, these residual pools of pERK might be sufficient to promote lytic gene expression. To test this idea, we probed immunoblots of infected siERK-treated RhF lysates for pERK (). In controls, we consistently detected more pERK2 than pERK1 even after correcting (see Materials and Methods) for the approximately 3:1-greater sensitivity of the pERK antibody to pERK2 compared to pERK1 (). With ERK1 knockdown efficiencies of close to 85% (, lane 2), we found that the average amount of remaining pERK1 was only ~11% of control levels, while levels of pERK2 were actually slightly (1.22-fold) higher than control levels, but this increase did not reach statistical significance ( and , lane 2). In contrast, despite >85% knockdown in ERK2 (, lane 3), residual pools of pERK2 remained at ~35% of the control levels (, lane 3). Furthermore, the level of pERK1 rose to nearly double that in the control cells but this increase was shy of statistical significance. Finally, with dual knockdown of ERK1 and ERK2, at 87% and >80% efficiency, respectively (, lane 4), the residual levels of the corresponding activated isoforms were 30% (for pERK1) and 48% (for pERK2) of their control values ( and , lane 4). These data show that pERK2 pools were maintained under all siERK conditions and were likely sufficient to drive lytic gene expression and viral production ( to ).
Fig 6 Residual pools of pERK2 remain following the siRNA knockdown of ERK isoforms. (A) Representative blot of cell lysates from siERK experiment () probed with anti-pERK antibody. (B) Relative changes in the levels of pERK1 and pERK2 shown graphically, (more ...) ERK1 acts as a negative regulator of viral production.
Although knockdown of the expression of ERK1, ERK2, or both during RRV infection led to only minor changes in the intracellular levels of viral structural proteins, this approach did not measure virion formation, release, or infectivity. We reasoned that rapid egress and release of assembled virions would minimize potential differences in intracellular viral protein levels affected by changes in one or more of the ERK isoforms. Due to the preferential activation and incorporation of ERK2 in RRV, we initially hypothesized that decreased levels of ERK2 might result in a decrease in overall viral production. To this end, we harvested viral particles from the media of the siRNA experiments shown in . We loaded particles derived from equivalent volumes of media and then separated the proteins in each sample by SDS-PAGE and probed the immunoblots for structural proteins, MCP, ORF45, and SCIP. We set the control level of each protein to 1.0 ( and , lane 1) and quantified the changes in protein levels from all other conditions relative to these controls. Remarkably, cells treated with siERK1 gave rise to markedly higher levels of viral particles, as evidenced by parallel elevations in the levels of all three structural viral proteins in the samples we collected from the media following centrifugation through a sucrose cushion (, lane 2). In contrast, siERK2 knockdown led to few to no discernible differences in particle accumulation in the media (, lane 3). Cells treated with the dual-ERK knockdown displayed a modest increase in the level of each viral protein (, lane 4). displays a graphical representation of these data from three independent experiments. Although the overall pattern of particle release remained consistent among the different siRNA conditions, the levels of released particles showed appreciable interexperimental variability. Since the number of MCPs per particle is invariant (955 copies) and since the MCP comprises the structurally resilient capsid, we measured this protein in a total of 7 experiments. We found that levels of particle-associated MCP in the media from cells treated with siERK1 and with siERK1 plus siERK2 were 6- and 1.5-fold greater than in the control media, respectively (). We also noted that under each of the siRNA conditions, the relative stoichiometries of ORF45 and SCIP per particle (normalized to MCP) did not change appreciably (), suggesting that the overall structural and compositional integrity of the virions was conserved.
Fig 7 Knockdown of cellular ERK1 markedly increases levels of virions released into the media. Equal volumes of media from the siERK experiments () were harvested and cleared of cells and debris. Viral particles were formed into pellets through a 35% (more ...)
Thus, in contrast to our initial hypothesis that ERK2 levels would play the dominant role in viral production and release, it appeared that ERK1 was perhaps the most critical modulator of these processes and suggested its role as a potent negative regulator. Nevertheless, lowering intracellular levels of ERK2 as well as ERK1 greatly dampened the overproduction of particles evident with ERK1 knockdown alone, suggesting that a minimal level of ERK2 is necessary for maximal virion production.
Intravirion ERK content reflects intracellular ERK expression.
Although the levels of ERK were suppressed within siERK-treated infected cells ( and ), the overall size and shape of the particles released under each condition appeared grossly similar to those in control samples (e.g., sucrose gradient-fractionated particles demonstrated structural protein peaks in parallel fractions; data not shown). In addition, the approximate stoichiometries of the tegument protein ORF45 and the capsid protein SCIP within the particles were similar regardless of the specific siERK condition (). However, it was unclear whether the intracellular ERK manipulations would lead to qualitative or quantitative disturbances in the incorporation of ERK in released virions. We reasoned that if any one of the ERK isoforms (or its corresponding activated form) were an essential structural component of the virus, the virions would incorporate similar numbers of ERK molecules. To approach this issue, we first probed virions produced under each of the siRNA conditions with antibody to total ERK and normalized each value to MCP in each sample, as we have described above. Virions released from cells treated with control siRNA had approximately 4-fold more ERK2 than ERK1 ( and , lane 1). Those from siERK1-treated cells contained almost exclusively ERK2, with only trace amounts of ERK1, and the amount of ERK2 was nearly double the amount in controls ( and , lane 2). Particles from siERK2-treated cells contained mostly ERK1 and only ~5% of the amount of ERK2 present in controls ( and , lane 3), while those from dual-siERK-treated cells contained slightly more ERK2 than ERK1 and only ~10% of the ERK2 present in the controls ( and , lane 4). Although the levels of ERK1 also differed in virions collected under each siERK condition, their differences from controls did not reach statistical significance ().
Fig 8 Intravirion ERK content reflects intracellular expression of ERK isoforms. (A) Representative blot of virions isolated from the media of RRV-infected cells treated with siRNA () and probed with anti-total ERK antibody. (B) Graphical representation (more ...)
Though these data demonstrated that the intravirion total ERK content approximated the relative levels of the intracellular ERK isoform(s) following siRNA manipulations (compare and ), it was still possible that proper assembly, structural integrity, or even infectivity might, instead, depend on the directed incorporation of pERK1 or, more likely, in light of our virion composition data, pERK2. If such a condition were critical for any of these or other unknown functions, we predicted that released infectious virions would likely preserve approximately equivalent amounts of pERK2 even in the face of siRNA perturbations in intracellular ERK levels. To address this possibility, we probed immunoblots of the virions from each siRNA condition for pERK ( and ). Control virions contained mainly pERK2, with minor amounts of pERK1 ( and , lane 1). Virions from siERK1-treated cells contained only trace amounts of pERK1 but amounts of pERK2 approximately equivalent to those seen with controls ( and , lane 2). The complementary knockdown with siERK2 led to released virions with 85% less pERK2 than in controls ( and , lanes 3 and 1). In contrast, the amount of pERK1 in these virions was essentially unchanged ( and , lanes 3 and 1), even though intracellular pERK1 levels more than doubled compared to the levels in control cells (, lanes 3 and 1). Finally, we noted that these virions contained approximately equal amounts of the two pERK isoforms ( and , lane 3) even though intracellular levels of pERK2 were nearly 2.5-fold lower than of pERK1 (, lanes 1 and 3). Virions released from dually siERK-treated cells had levels of pERK1 and pERK2 that were lower than in control virions by ~82% and ~86%, respectively, but pERK2, again, remained the dominant species ( and , lane 4).
Alterations in intravirion ERK content maintain or enhance virion infectivity.
We next tested whether the perturbations in the intracellular ERK content that led to differing amounts of particle production ( and ) might also affect viral infectivity. Since the various ERK knockdowns also affected intravirion ERK content (), we also tested whether the relative infectivities of virions in culture might change as a consequence of either quantitative or qualitative differences in their ERK content. We first determined the viral titer of the media from the infected RhF following knockdown of ERK1, ERK2, or both and compared these conditions to the control (). To ensure that siRNA treatment did not negatively affect the efficiency of viral infection, we infected cells with or without control siRNA. We found no statistically significant difference in the number of infectious particles released from cells treated with or without control siRNA (2.8 ± 1.2 × 107 compared to 2.9 ± 1.2 × 107 PFU/ml, respectively). In contrast, compared to siRNA-treated controls, the titer in the media from the ERK1 knockdown condition was significantly greater, with an increase of 12.9-fold ± 5.0-fold relative to controls (, columns 1 and 2), suggesting, at a minimum, that the large number of particles released following ERK1 knockdown () retained their infectivity. (Note that, although the absolute titers determined between repetitions of these experiments showed biological variability [e.g., 1.92 × 10E6 to 1.44 × 10E7 PFU/ml for siCNL], the patterns of effects of the different siRNA conditions within each experiment were remarkably consistent.) Though the titers from the ERK2 and dual-knockdown conditions were slightly higher than the controls, these differences did not reach statistical significance (, columns 3 and 4).
Fig 9 Alterations in intravirion ERK content maintain or enhance virion infectivity. (A) Viral titers in the media of RRV-infected RhF treated with the indicated siRNA were determined in duplicate. For each experiment, relative titers were calculated by dividing (more ...)
In separate experiments, we also compared titers to viral genome equivalents in the media from each siRNA condition. These experiments, again, showed that the ERK1 knockdown condition gave the highest titer (though it was only 4.5-fold higher in this series) compared to the control siRNA condition. However, surprisingly, the qPCR measurements of genome equivalents were not significantly different for any of the conditions (siCNL, 1.0 × 107; siERK1, 8.4 × 106; siERK2, 1.3 × 107; and siERK1/2, 8.3 × 106 genome equivalents/ml). Since differences in the encapsidated DNA content did not mirror the increased titer in the media from the ERK1 knockdown condition (at least within the precision of qPCR), these data suggested that ERK1 knockdown may lead to the production of particles with higher infectivity.
As we discussed above, the intensity of the MCP signal on a quantitative immunoblot of virions correlates well with particle number. We exploited this linear relationship to calculate the relative amounts of MCP in each sample, allowing us to normalize the titers to the relative numbers of particles. The titers () and corresponding MCP immunoblot signals () represented parallel analyses of sister aliquots in each experiment. For each experiment, we divided the absolute titer under each condition by its corresponding MCP value and compared each to the control, which we set to 1.0 (, column 1). Particles from the siERK1 condition (, column 2) were slightly more than 3 times more infectious than control particles. In addition, virions produced from the siERK2 condition (, column 3) were also more infectious (at almost 2 times greater than the control level). The infectivity of virions from the dual-siERK condition, however, was not significantly different from those from control samples ( and , columns 4). We noted that the viral species that were most infectious (those from siERK1) contained approximately twice as much ERK2 as and significantly less pERK1 than the control, whereas the only distinguishing component of virions from siERK2 was that these virions maintained the levels of pERK1 evident in control virions. Thus, it seems that the amount and level of activation of the ERK isoforms were not sufficient to predict these modest differences in relative infectivity of the virions. In the end, the data argue that RRV can tolerate dramatic reductions in both total and activated ERK isoforms as well as variability in the relative abundances of these species without a significant loss in infectivity.
Biased incorporation of ERK2 and pERK2 into RRV particles.
We noted that the relative amounts of ERK1 and ERK2 as well as pERK1 and pERK2 within virions generally paralleled their relative abundances in the infected RhF under each siERK condition (compare with 5B and 8D with 6B, respectively). However, it was also evident that virions contained a disproportionate amount of ERK2 and pERK2, suggesting a bias toward incorporation of these species in preference to ERK1 and pERK1, respectively, during virion assembly. To more precisely evaluate this potential selectivity, we determined the ratio of ERK2 to ERK1 as well as pERK2 to pERK1 in cells and virions under each siERK condition.
In RRV-infected cells treated with nontargeting siRNA (siCNL), we found that the ratios of ERK2 to ERK1 and pERK2 to pERK1 were 1.81 ± 0.26 and 2.61 ± 0.38, respectively; however, the corresponding ratios were consistently and markedly higher in virions (7.92 ± 2.38 and 7.71 ± 1.0, respectively) (, upper and lower panels, first column sets). This bias favoring the packaging of ERK2 (and pERK2) in newly formed virions remained evident following marked reductions in intracellular ERK1 levels ( and , lane 2). In this setting, the ratio of ERK2 to ERK1 and pERK2 to pERK1 in cells rose to 10.2 ± 1.2 and 26.8 ± 5.0, respectively. Nevertheless, the virions released from these cells showed ratios that were, again, even higher (29.3 ± 5.1 and 64.35 ± 7.8, respectively) (, upper and lower panels, second column sets).
Fig 10 Biased incorporation of ERK2 and pERK2 in RRV virion assembly. Using data from , , and , involving 4 independent experiments, we determined ratios of ERK2 to ERK1 (upper panel) and pERK2 to pERK1 (lower panel) in cells (more ...)
The total ERK2 bias, however, was no longer evident following the ERK2 or dual knockdown in the cell (, upper panel, third and forth column sets). The ratios of ERK2 to ERK1 in the virions and cells were statistically indistinguishable in the ERK2 knockdown (0.14 ± 0.05 and 0.18 ± 0.04, respectively; , upper panel, third column set). In the double knockdown, the ratios were somewhat higher in both the virions and cells but, again, the values determined for virions and cells were statistically indistinguishable (1.91 ± 0.47 and 2.22 ± 0.39, respectively; , upper panel, fourth column set). In contrast, the virions continued to display a strong incorporation bias for pERK2 in preference to pERK1 even in the setting of low relative abundance of intracellular pERK2 (siERK2). The ratio of pERK2 to pERK1 was 1.40 ± 0.34 in virions but only 0.36 ± 0.14 in cells (, lower panel, third column set). This trend continued for ratios of pERK2 to pERK1 in the virions from the double knockdown, but the difference did not reach statistical significance (9.99 ± 4.12 and 4.61 ± 0.88 for virions and cell, respectively). Taken together, these data suggested that, compared to the intracellular milieu from which these virions derived, released virions appeared to preferentially incorporate total but, particularly, activated ERK2 (pERK2) over the corresponding ERK1 species by a margin of 3 or 4 to 1.