F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cell's ubiquitin-proteasome machinery.

The ecology of highly pathogenic avian influenza (HPAI) H5N1 has significantly changed from sporadic outbreaks in terrestrial poultry to persistent circulation in terrestrial and aquatic poultry and potentially in wild waterfowl. A novel genotype of HPAI H5N1 arose in 1996 in southern China and through ongoing mutation, reassortment, and natural selection, has diverged into distinct lineages and expanded into multiple reservoir hosts. The evolution of Goose/Guangdong-lineage highly pathogenic H5N1 viruses is ongoing: while stable interactions exist with some reservoir hosts, these viruses are continuing to evolve and adapt to others, and pose an un-calculable risk to sporadic hosts, including humans.

Tumor necrosis factor alpha (TNF-␣) activates the nuclear factor B (NF-B) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-B activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-B activation by TNF-␣. Immunofluorescence analyses revealed that nuclear translocation of NF-B subunit p65 protein in TNF-␣-treated HeLa cells was blocked by CP77. CP77 did so without blocking IB␣ phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-B subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-B activation. We thus propose a model in which poxvirus CP77 suppresses NF-B activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-B subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-B modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-B did not relieve the growth restriction of VV-hr-GFP in HeLa cells.Vaccinia virus, the prototype of the poxvirus family, infects a wide range of cells in vitro and animal species in vivo (14). Vaccinia virus has a double-stranded DNA genome that encodes 263 open reading frames (ORFs). Vaccinia virus expresses different classes of viral genes in a cascade-regulated manner and completes the virus life cycle in the cytoplasm of infected cells (11).To replicate successfully in infected hosts, poxviruses have evolved various strategies to overcome cellular immune responses (20, 39). Viral infections activate cellular antiviral signaling and inflammatory responses (49), such as NF-B, which plays a critical role in inflammatory signaling and immune activation (23). NF-B contains five different members, NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65), RelB, and c-Rel, all of which share a Rel homology domain for DNA binding, dimerization, and interaction with IB (22, 23). The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit (16,26). In the inactive state, dimerized NF-B (such as p65/p50) is bound by IB␣, and the crystal structure of the IB␣/p65/p50 complex shows multiple contact sites between the ankyrin repeats of IB␣ and NF-B (29). In well-characterized canonical NF-B signaling, such as tumor necrosis factor alpha (TNF-␣) treatment, receptor activation sends intracellular sig...

Ankyrin-repeat (ANK) protein-interaction domains are common in cellular proteins but are relatively rare in viruses. Chordopoxviruses, however, encode a large number of ANK domaincontaining ORFs of largely unknown function. Recently, a second protein-interaction domain, an F-box-like motif, was identified in several poxvirus ANK proteins. Cellular F-box proteins recruit substrates to the ubiquitination machinery of the cell, a putative function for ANK/poxviral F-box proteins. Using publicly available genome sequence data we examined all 328 predicted ANK proteins encoded by 27 chordopoxviruses that represented the eight vertebrate poxvirus genera whose members encode ANK proteins. Within these we identified 15 putative ANK protein orthologue groups within orthopoxviruses, five within parapoxviruses, 23 within avipoxviruses and seven across members of the genera Leporipoxvirus, Capripoxvirus, Yatapoxvirus, Suipoxvirus and Cervidpoxvirus. Sequence comparisons showed that members of each of these four clusters of orthologues were not closely related to members of any of the other clusters. Of these ORFs, 67 % encoded a C-terminal poxviral F-box-like motif, whose absence could largely be attributed to fragmentation of ORFs. Our findings suggest that the large family of poxvirus ANK proteins arose by extensive gene duplication and divergence that occurred independently in four major genus-based groups after the groups diverged from each other. It seems likely that the ancestor ANK proteins of poxviruses contained both the N-terminal ANK repeats and a C-terminal F-box-like domain, with the latter domain subsequently being lost in a small subset of these proteins. INTRODUCTIONChordopoxviruses, a diverse subfamily of the family Poxviridae of DNA viruses, infect a wide range of vertebrates (Moss, 2007). Terminal regions of their large linear genomes typically encode host regulating factors and multiple ankyrin-repeat (ANK) proteins (Damon, 2007). The ANK protein-interaction domain consists of two or more ankyrin motifs and is frequently found in cellular proteins, but relatively rarely in virally encoded proteins, with chordopoxviruses a notable exception (Bork, 1993;Li et al., 2006;Mosavi et al., 2004).The only classified chordopoxvirus not encoding ANK proteins is molluscum contagiosum virus, the sole member of the genus Molluscipoxvirus. Poxviruses of the remaining eight genera each encode numerous ANK proteins (Afonso et al., 2000(Afonso et al., , 2005 Cameron et al., 1999;Chen et al., 2003;Delhon et al., 2004;Gubser & Smith, 2002;Tulman et al., 2001Tulman et al., , 2002Tulman et al., , 2004Tulman et al., , 2006. Specific ANK proteins of cowpox virus (CPX), vaccinia virus (VACV) and myxoma virus (MYXV) have been shown to affect the viruses' host range and virulence, but the functions of the vast majority of poxvirus ANK proteins remain unknown (Bradley & Terajima, 2005;Hsiao et al., 2004 Hsiao et al., , 2006Perkus et al., 1990;Ramsey-Ewing & Moss, 1996). Recently, a second proteininteraction domain, a truncated F-b...

Avian-origin influenza A(H7N9) recently emerged in China, causing severe human disease. Several subtype H7N9 isolates contain influenza genes previously identified in viruses from finch-like birds. Because wild and domestic songbirds interact with humans and poultry, we investigated the susceptibility and transmissibility of subtype H7N9 in these species. Finches, sparrows, and parakeets supported replication of a human subtype H7N9 isolate, shed high titers through the oropharyngeal route, and showed few disease signs. Virus was shed into water troughs, and several contact animals seroconverted, although they shed little virus. Our study demonstrates that a human isolate can replicate in and be shed by such songbirds and parakeets into their environment. This finding has implications for these birds’ potential as intermediate hosts with the ability to facilitate transmission and dissemination of A(H7N9) virus.