Here, we report a novel function for the vaccinia K1 protein; it inhibits PKR activation. Since active PKR attenuates protein synthesis, one attractive hypothesis was that the K1 protein, in preventing PKR activation, would allow for virus replication in RK13 and HeLa cells. This model was bolstered by the observation that viruses that allowed PKR or eIF2α activation were not replication competent in HeLa or RK13 cells (Meng and Xiang, 2006
), respectively. While PKR-deficient HeLa cells indeed allowed for a partial restoration of viral protein synthesis, these cells remained unable to support the synthesis of the late L1 protein or the replication of S2C#2. Thus, K1’s PKR inhibitory phenotype only partially contributes to permissivity in HeLa cells. One conclusion from this study is that there are PKR-independent mechanisms for controlling viral protein synthesis during infection. For example, for translation to occur, capped mRNA must be complexed with eIF4F, a complex that is formed only upon the inactivation of eIF4E binding proteins. Vaccinia virus infection inactivates the 4E-BP1 binding protein to promote eIF4F complex formation. Whether this event is critical to the host-range phenotype is unknown (Walsh et al., 2008
The E3 and K3 proteins are expressed early in infection, and each inhibit PKR activation (Rivas et al., 1998
; Romano et al., 1998
; Sharp et al., 1998
; Shors et al., 1997
). ΔK1L infection of RK13 cells results in a dramatic decrease in early gene translation (Ramsey-Ewing and Moss, 1996
), which would presumably include E3 and K3 protein synthesis. Thus, an initial concern was that the ΔK1L effect in these rabbit cells was indirect, due to a lack of E3 and K3 proteins. However, we found that E3 proteins were expressed in RK13 cells, regardless of the absence of the K1L ORF. Further examination revealed that other early proteins, such as the M2 and J3 products, were also detected in ΔK1L-infected RK13 cells (data not shown), suggesting that the K3 protein would also be synthesized during infection as well. Since early protein synthesis, as measured by the 35
S-methionine metabolic labeling of nascent proteins during virus infection, was detected for at least one hour after ΔK1L infection of RK13 cells (Ramsey-Ewing and Moss, 1996
), our results were not surprising. Most important, these data indicate that the PKR activation phenotype in ΔK1L–infected RK13 cells is not indirect; i.e., PKR activation in ΔK1L–infected cells is not simply due to a lack of E3, or other known or undiscovered, PKR inhibitory proteins. For HeLa cells, infection with viruses lacking both the K1L and C7L ORFs results in a block in intermediate gene translation (Hsiao et al., 2004
). Thus, E3 (and K3) proteins are expected to be synthesized, an event we detect in our system.
We identified that the ANK2 repeat of K1 is responsible for PKR inhibitory function, but cannot rule out that other K1 ANK repeats may also possess inhibitory function. Given that other ANK repeats of K1 are important for its host-range function (Meng and Xiang, 2006
), one prediction is that mutations in other ANK repeats would be expected to affect K1’s PKR inhibitory function. We purposefully limited our studies to the ANK2 region since the smallest number of amino acid substitutions was made in this repeat (Meng and Xiang, 2006
). Further, two of these constructs (S2C#2 and S2C#3) yielded different host range phenotypes, characteristics that would enable us to easily assess if PKR activity and host range phenotype were related.
ANK repeats are 33 residues in length, and the motif is defined as a β-hairpin-helix-loop-helix structure (Sedgwick and Smerdon, 1999
). These motifs are found in proteins in nearly all phyla, and have myriad functions (Sedgwick and Smerdon, 1999
). Diversity in binding partners for ANK-containing proteins is attained by variation in the surface residues of ANK repeats, and by the number of ANK repeats stacked together to form a stable structure (Sedgwick and Smerdon, 1999
). Substitution mutations were originally made in the ANK2 region that would only affect surface residues, affecting potential binding site for K1 interacting partners, but allowing for the ANK repeat to keep its structure, since structure is likely important for its ability to interact with its target protein (Meng and Xiang, 2006
). The mutated residues in S2C#2 lie in the outer helix, a potential surface for protein-protein interactions, while the mutations in S2C#3 lie downstream of the outer helix (Meng and Xiang, 2006
). Thus, the finding that the S2C#2 virus allowed for PKR activation likely reflects a loss of binding to a target protein. A previous study identified a rabbit homolog of the human cellular ACAP2 protein that interacts with K1 protein in vaccinia-infected RK13 cells (Bradley and Terajima, 2005
). Whether K1-ACAP2 interactions are critical for its ability to inhibit PKR activity is an area of future research. It was noticed that the ANK2 regions responsible for PKR inhibition were different for RK13 versus HeLa cells. For RK13 cells, residues in both the N and C terminal portion of ANK2 were necessary for inhibitory function, while residues only in the C terminal portion of ANK2 were important for HeLa cells. These differences may indicate that the K1 protein interacts with a different subset of rabbit versus human proteins.
While activated PKR is best known for its anti-viral effect of inhibiting protein synthesis, it possesses other biological properties that neutralize virus infections (Garcia, Meurs, and Esteban, 2007
). For example, PKR also induces apoptosis, a rapid form of cell death that eliminates virus-infected cells (Lee and Esteban, 1994
). Whether the K1 protein would prevent PKR-induced apoptosis is unknown. PKR also activates the NF-κB transcription factor (Maran et al., 1994
; Yang et al., 1995
). Since this cellular transcription factor induced the expression of anti-viral immune molecules, this property of PKR could also inhibit virus replication. To date, there are multiple vaccinia proteins that inhibit PKR activity, including K1, C7, E3 and K3 (Garcia, Meurs, and Esteban, 2007
). For this model system, in which HeLa cells are used, it does not appear that the E3L or the K3L ORF, both of which are present and presumably expressed during virus infection, are involved in virus-mediated PKR activation. These data would argue that molecules in addition to dsRNA, including PACT and Mda7, activate PKR during poxvirus infection. Furthermore, the presence of myriad vaccinia proteins likely reflect the fact that different insults activate PKR.
The CP77, K1 and C7 proteins can functionally complement each other to allow for a productive vaccinia infection in HeLa cells (Perkus et al., 1990
). These three proteins also prevent PKR activation (Meng, Chao, and Xiang, 2008
) (Hsiao et al., 2004
). The K1 and CP77 proteins both possess ankyrin repeats. The CP77 protein also has an F-box motif (Chang et al., 2009
), a motif lacking in the K1 protein. In contrast, no obvious F-box or ankyrin motifs are present in C7 proteins. One prediction, then, is that the CP77 and K1 proteins will utilize similar molecular mechanisms to alter PKR activation, while the C7 product will likely utilize a mechanism distinct from CP77 or K1. As shown here, the K1 protein prevents PKR activation, as measured by an inhibition of PKR phosphorylation. The K1 protein lacks an obvious dsRNA binding motif, making it unlikely that K1 functions like the E3 protein, binding to dsRNA as its mechanism to inhibit PKR activation. A simple explanation for K1’s molecular mechanism is that the K1 protein binds to PKR, preventing its dimerization and subsequent auto-phosphorylation. Ankyrin repeats, like the ones present in K1, are important for mediating protein-protein interactions (Bork, 1993
), making it reasonable to suggest that the K1 protein could form a stable complex with PKR. A second possibility is that the K1 protein indirectly inhibits PKR activation by binding to cellular PKR activating proteins, such as PACT and Mda7 (Garcia, Meurs, and Esteban, 2007
; Zhang et al., 2009
). Thus, future directions include assessing K1 interactions with the above proteins and assessing if K1 binding function correlates with PKR inhibition.