Successful replication of poxviruses in many cell types depends on virus-encoded host range proteins functioning downstream of viral entry (14
). Since the host range proteins are dispensable for viral replication in some cell types, they presumably play no direct role in virion biogenesis but either provide an essential function that is lacking in a particular cell type or play an essential role in downmodulating certain cell type-specific antiviral responses. Studying the functions of the host range proteins thus provides an avenue for discovering critical innate antiviral responses as well as viral strategies of evading these responses. However, the host range proteins are among the least understood in the poxvirus proteome (14
). In the present study, we solved the crystal structure of VACV K1, representing the first structure for any poxvirus host range proteins. The K1 structure provides a structural perspective for our previous and current mutagenesis studies of K1 (17
). Collectively, the structure function studies of K1 offer insights into the molecular mechanisms of the host range proteins.
The structure of K1 shows that K1 consists entirely of ANK repeats (Fig. ), suggesting that K1 functions solely through ligand interaction, since ANK repeats are only known for a role in ligand interaction (13
). The structure also confirms that K1 has no F box, which is present in the majority of poxvirus ANK repeat proteins but was predicted not to be present in K1 (18
). ANK repeat proteins form the largest group of proteins encoded by the poxviruses (28
). Many poxvirus ANK repeat proteins are involved in regulating the host range or modulating host signaling pathways (3
). A unique feature for many poxvirus ANK repeat proteins is the presence of a C-terminal F-box, which binds to core elements of the ubiquitin ligase complex (28
). It was proposed that poxvirus ANK-F-box proteins modulate diverse cellular pathways by targeting different cellular proteins for ubiquitination (18
). The ANK repeats presumably bind to specific cellular targets, which are ubiquitinated by the ubiquitin ligase complex that are brought in by the F-box. This, however, is unlikely to be the mechanism of action for K1, since there is no F-box in K1 structure. Similarly, although a functional F-box is present in the host range proteins CP77 and 68k-ank, the F-box is not required for their host range function (3
The K1 structure provides a much-needed structural perspective on previous and current mutagenesis study of K1. Prior to the elucidation of the atomic-level structure of K1, we performed structure function studies of K1 (17
). To avoid introducing mutation that affects structure, we substituted the K1 ANK repeats with a well-defined artificial ANK repeat. The substitution presumably changed the surface residues of K1 ANK repeats without altering the overall structure, since different ANK repeats fold into almost identical structure with only the surface residues that are unique (13
). With this approach, we were able to identify Phe82 and Ser83 as residues that are important for K1's function (17
). However, it remained possible that the substitution of these two residues might impair K1's host range function through indirect effect on protein structure. Now with the determination of K1 structure, we are able to exclude this possibility, since these two residues are on the surface of the molecule (Fig. ), playing no structural role. Similarly, once we identified Cys47 and Asn51 as critical residues for K1's function in the present study, we could see from the structure that these residues are also solvent exposed and unlikely to play any structural role (Fig. ). On the other hand, when the substitution of Tyr35, Tyr36, and Ile38 were found to disrupt K1's function and reduced the K1 protein level, we could see from the structure that these residues are involved in inter-repeats interaction and conclude that the substitutions of these residues disrupt K1's function by destabilizing the protein (Fig. ).
The K1 residues that were found to be essential for the host range function center on a contiguous surface (Fig. ), suggesting that they are part of a binding surface for some ligand. Cys47 and Asn51 are separated in primary sequence from Phe82 and Ser83, but they are spatially adjacent to each other, forming a contiguous surface. We believe that this surface is the binding surface for some protein factor and that the binding free energy is distributed over these four residues. This is probably why substitution of one of these four residues only cause partial defect in the host range function, whereas substitution of more than one residues causes a more severe defect. Previously, we found that substituting Phe82 or Ser83 caused partial defect in the replication of VACV in HeLa cells but that substituting both residues completely abolished viral replication in HeLa cells (17
). In the present study, we found that substituting Cys47 only caused minor defect in replication of VACV in RK13 cells, whereas substituting Asn51 caused a more severe defect (Fig. ). Substituting both residues, along with two other nonessential residues, caused a much more severe replication defect in RK13 and HeLa cells.
All four K1 residues that are critical for the host range function located on the convex back surface of the molecule (Fig. ), suggesting that K1 uses a novel model for ligand interaction. In previously studied protein complexes involving ANK repeats, the concave surface is found to bind the target molecules (13
). The concave surface consists of stacked α-helices and β turns, at nearly 90° angle to each other (Fig. ). The L-shape concave surface could enclose the ligand with two perpendicular surfaces, therefore providing a much tighter binding than the flat back surface could. It is possible that K1 may only need to interact with its cellular target transiently to exert its effect, so the convex back surface of K1 may provide sufficient binding affinity. This may be why we have not been able to isolate any biologically relevant binding partners for K1 by using the affinity purification method. ACAP2, a GTPase-activating protein for ARF6, is the only cellular protein found to interact with K1 (2
). However, we showed previously that substitution of both Phe82 and Ser83 abolished the host range function but had no impact on ACAP2 binding (17
). Similarly, we found that substituting both Asn51 and Cys47 (in mut6) impaired the host range function but had no effect on ACAP2 binding (data not shown), further supporting that ACAP2 binding is not sufficient for K1's host range function. In addition, we showed previously that the substitutions at ANK5 disrupted ACAP2 binding with no adverse effect on K1's host range function, indicating that ACAP2 binding is also not necessary for the host range function (17
). K1 may interact with ACAP2 to affect some cellular processes that are not directly related to the host range restriction, and it may do so with the canonical concave surface.
On the convex surface adjacent to the four critical K1 residues, we noticed a large positively charged surface patch comprised of at least six residues (Arg44, Lys75, Lys78, Lys111, Lys115, and Lys116) (Fig. ). This surface patch, however, does not appear to be involved in specific charge-charge interactions, since reversing the charge of some of the residues did not affect K1's host range function. The substitution of both Lys75 and Lys78 with Glu was previously shown to have no effect on K1's host range function (17
). In the present study, we showed that Arg44Glu substitution (in mut5) also had no effect on K1's function (Fig. ). The four critical K1 residues are clustered in a nearby relatively hydrophobic region (Fig. ), suggesting that K1-ligand recognition could be rather hydrophobic in nature rather than through charge-charge interactions.
FIG. 4. Electro-potential of K1 surface. (A) Top view; (B) side view. The positively and negatively charged surfaces are colored blue and red, respectively. Notice the large positively (blue) charged patch on the surface of ANK1-4. The relatively hydrophobic (more ...)
Some of our K1 mutations showed different effects on VACV replication in RK13 cells than in HeLa cells. mut8 and mut9, which have substitution in Asn51 of K1, failed to replicate in RK13 cells but replicate efficiently in HeLa cells (Fig. ). mut9 and mut10, which have substitution in Cys47 of K1, replicated with a decreased efficiency in RK13 cells but replicated normally in HeLa cells. Only when both Asn51 and Cys47 were substituted did we observe a defect in replication in both HeLa and RK13 cells. The reason for this discrepant effect of K1 mutations in different cells is unknown, but this is consistently with our previous mutagenesis study of K1 (17
). For example, while individual substitution of Phe82 or Ser83 completely abolished viral replication in RK13 cells, it only reduced VACV replication in HeLa cells. RK13 cell is known to be a unique cellular host for VACV. Although both K1 and C7 can support viral replication in many human and murine cell lines, only K1 can do so in RK13 cells (24
). We suspect that the host factor targeted by K1 may exist at a higher concentration or with a higher potency in RK13 cells than in HeLa cells, so mutations that slightly decrease the affinity of K1 to this host factor may be able to affect VACV replication in RK13 cells but may not be sufficient to affect VACV replication in HeLa cells. Therefore, RK13 cells serve as a more sensitive host for assessing K1 mutations, allowing us to detect mutations that individually have a minor effect but collectively have a major effect on K1's function.
Both K1 and CP77 were previously reported to inhibit the activation of NF-κB in response to VACV infection (3
). However, this function of the host range proteins is not responsible for alleviating the host range restriction of a VACV host range mutant with no K1L and C7L (VV-hr). Chang et al. reported previously that a mutated CP77, which failed to inhibit NF-κB, was still able to rescue the growth restriction of VV-hr in HeLa cells (3
). Conversely, knocking down NF-κB protein expression in HeLa cells failed to relieve the growth restriction of VV-hr (3
), suggesting that inhibition of host NF-κB activation is neither necessary nor sufficient for rescuing host range restriction of VV-hr. Our unpublished data indicate that, if C7L is absent, K1L is necessary for VACV to inhibit host NF-κB activation in HeLa cells. However, which K1 residues are critical for NF-κB inhibitory function remain to be determined. More recently, we found that K1 and C7 antagonize antiviral activities induced by type I interferons (16
). K1 residues that are critical for its host range function were found to be also essential for its ability to antagonize type I interferons (16
), suggesting that the interferon-induced antiviral factor that is targeted by K1 is the same factor that restricts the growth of ΔK1LΔC7L VACV mutant in nonpermissive cells.