The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drugresistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase II␤. One can prevent the interaction by introducing a C 11 -to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase II␣/␤ in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase II␣ and II␤ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases II␣/␤ in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.Topoisomerases play a critical role in modulating DNA superhelical density and in unknotting and decatenating the structures formed during replication, recombination, and repair (reviewed in references 2 and 5). Mammalian cells carry several different kinds of topoisomerases, classified as being either type I or type II enzymes. Both classes of enzyme catalyze DNA strand breakage and rejoining, but type I enzymes catalyze reactions involving single-strand breaks, while the type II topoisomerases catalyze double-strand cleavage reactions. The mammalian type IB enzyme (Topo I) is well adapted for removing the superhelical stress created by the replication and transcription machinery. The two closely related type IIA enzymes (Topo II␣ and Topo II␤) may also catalyze these same reactions but probably play a more critical role in processes like chromatin reorganization and chromosome segregation. A third class of type IA enzymes (Topo III␣ and Topo III␤)

Section: Properties Of Etoposide-resistant Virusesmentioning

confidence: 99%

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Lin¹,

Li²,

Irwin³

et al. 2008

“…Bars, 5 m. Viral factories are highlighted with dashed lines. Note: I3L, previously shown to be expressed early in infection and localize to viral factories (28,40), was used as a marker for the infected cells and viral factories.…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cells infected with VV (WR strain) were fixed at 3, 6, 9, 12, and 24 hpi and processed for immunostaining with antibodies to golgin-97 and the VV single-stranded DNA-binding protein, I3L, which is expressed early in infection (28,36). Because I3L was previously shown to localize to the viral DNA replication sites (28,40), antibodies to this protein were used as a marker for the viral factories, in particular early in infection when viral DNA (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A trans -Golgi Network Resident Protein, golgin-97, Accumulates in Viral Factories and Incorporates into Virions during Poxvirus Infection

Alzhanova

Hruby

2006

Poxviruses are the only DNA viruses known to replicate and assemble in the cytoplasm of infected cells. Poxvirus morphogenesis is a complicated process in which four distinct infectious forms of the virus are produced: intracellular mature virus, intracellular enveloped virus, cell-associated enveloped virus, and extracellular enveloped virus. The source of primary membrane wrapping the intracellular mature virus, the first infectious form, is still unknown. Although the membrane was suggested to originate from the endoplasmic reticulum-Golgi intermediate compartment, none of the marker proteins from this or any other cell compartments has been found in the intracellular mature virus. Thus, it was hypothesized that the membrane is either extensively modified by the virus or synthesized de novo. In the work described here, we demonstrate that a host cell protein residing in the trans-Golgi network membrane, golgin-97, is transported to the sites of virus replication and assembly and becomes incorporated into the virions during poxvirus infection. Inside the virion, golgin-97 is associated with the insoluble core protein fraction. Being able to adopt a long rod-like structure, the protein apparently extends through the virion envelope and protrudes from its surface. Here we discuss the potential role and functions of golgin-97 in poxvirus replication and propose two working models.Poxviruses are large enveloped double-stranded DNA viruses with a complex morphology. Vaccinia virus (VV), the most extensively studied member of the Poxviridae, possesses a genome of approximately 192 kbp in size that encodes over 200 proteins whose expression is temporally regulated (22). Unlike other DNA viruses, VV replicates and assembles in the perinuclear compartments within the cytoplasm, termed viral factories or virosomes (22). The process of virus morphogenesis is very complicated and occurs in several stages. It begins with the assembly of membrane crescents around electron-dense material containing viral DNA and core proteins (11), leading to formation of spherical immature virus (IV). This step is followed by a series of proteolytic cleavages and condensation of the virus core, transforming IV into oval or brick-shaped intracellular mature virus (IMV), the first infectious form of VV (21). IMV represents the majority of produced virions and remains trapped inside the cell until its lysis. A subfraction of IMV moves on microtubules from the viral factories to the trans-Golgi network (TGN) (30) to acquire two additional membranes and become intracellular enveloped virus (IEV). Transported to the cell surface along microtubule networks (5, 9, 27, 38), IEV particles bud through the plasma membrane and lose the outermost envelope. This process generates cellassociated enveloped virus (CEV). CEV induces formation of actin tails that propel the particles, now referred to as extracellular enveloped virions (EEV), outside the cell and facilitates cell-to-cell and long-range dissemination of the virus.The origin of the virion membran...

“…Cells were fixed with paraformaldehyde, permeabilized, and blocked with bovine serum albumin and fish gelatin as described previously (9). p28 was detected using fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody (M2; Sigma), rabbit anti-I3L was used to detect virus factories (16), and mouse monoclonal anti-HA (HA-7; Sigma) was used to visualize ubiquitin. Primary antibodies were detected with goat anti-rabbit Alexa Fluor 350 (Molecular Probes) and goat anti-mouse Rhodamine B (Biosource, Camarillo, Calif.), respectively.…”

mentioning

confidence: 99%

The Poxviral RING Protein p28 Is a Ubiquitin Ligase That Targets Ubiquitin to Viral Replication Factories

Nerenberg

Taylor

Bartee

et al. 2005

The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence.Poxviruses are large, complex viruses that replicate in the cytoplasm of infected cells (10). The poxviral RING (really interesting new gene) domain protein, p28, localizes to viral factories and is encoded by members of the leporipoxviruses and orthopoxviruses (12,14). Notable exceptions include vaccinia virus (VV) strain WR, which contains a truncated copy of p28, and VV strain Copenhagen, which lacks p28 entirely (12,14). Although the precise molecular function of p28 is presently unknown, p28 expression has been linked to apoptosis inhibition and a p28 knockout in ectromelia virus was strongly attenuated, resulting in reduced growth in macrophages and the complete clearance of virus from infected mice (2,3,11,12).Recently, a number of RING-containing proteins have been shown to act as ubiquitin ligases (RING-E3s) which simultaneously interact with a specific substrate and a ubiquitin-conjugating enzyme (ubc), or E2, to mediate substrate-specific ubiquitination (7). In vitro, RING-E3s catalyze the formation of polyubiquitin in the presence of ubiquitin, ubiquitin-activating enzyme (E1), E2, and ATP (8). Given the highly conserved RING domain in p28, we examined whether VV p28 and the myxoma virus (MV) homologue, M143R, display ubiquitin ligase activity in vitro. VV p28, derived from strain IHD-W (14), and M143R, from MV strain Lausanne, were fused to glutathione S-transferase (GST) and purified from Escherichia coli as described previously (9). Mutations in the RING domains of M143R and p28 were constructed by replacing two conserved cysteines, C173 and C176, with serine, which disrupts the ability of RING domains to complex with zinc and abolishes E3 ligase activity (9). Furthermore, the RING domain of p28 was deleted by truncation at residue 184, resulting in a construct consistent with the p28 truncation in VV strain WR. At a concentration of 3 M, each GST fusion was combined in vitro with 50 nM rabbit E1 (Boston Biochem), 0.5 M human E2 UbcH5a (gift of R. Everett and purified as described previously [1]), 10 mM ATP, and 28 M ubiquitin. Negative and positive controls consisted of GST and the RING domain of herpes simplex virus type 1 ICP0 (1), respectively. The in vitro ubiquitination reaction was performed in a solution of 20 l of 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM dithiothreitol for 90 min at 30°C, and high-molecular-weight (HMW) ubiquitin conjugate formation was determined by im...