Steven S. Broyles

Host Cell Nuclear Proteins Are Recruited to Cytoplasmic Vaccinia Virus Replication Complexes

ABSTRACT The initiation and termination of vaccinia virus postreplicative transcription have been reported to require cellular proteins, some of which are believed to be nuclear proteins. Vaccinia virus replicates in the cytoplasmic compartment of the cell, raising questions as to whether vaccinia virus has access to nuclear proteins. This was addressed here by following the fate of several nuclear proteins after infection of cells with vaccinia virus. The nuclear transcription factors YY1, SP1, and TATA binding protein were found to colocalize with virus replication complexes in the cytoplasm of infected cells. In addition, the nuclear proteins RNA polymerase II, TAFIIp32, and histone deacetylase 8, but not the structural protein lamin B, also were found in the cytoplasm of the cell. The association of YY1 with replication complexes was dependent on DNA replication and required only the DNA binding domain of the protein, indicating that DNA binding alone may be responsible for the association of nuclear transcription factors with viral replication complexes in the cytoplasm. The cytoplasmic localization of YY1 was resistant to the nuclear export inhibitor leptomycin B. Evidence is presented indicating that nuclear import and export pathways were not adversely affected by vaccinia virus infection. These observations indicate that vaccinia virus replication complexes have ready access to nuclear proteins by allowing leakage from the nucleus.

Transcription Factor YY1 Is a Vaccinia Virus Late Promoter Activator

Vaccinia virus has a DNA genome, yet replicates in the cytoplasmic compartment of the cell. We previously described the identification of a cellular protein having high affinity for vaccinia virus late promoter DNA. Sequence substitutions in the vaccinia I1L promoter were used to define a 5-nucleotide block at the transcription initiation site as essential for interaction with the protein. Within this sequence is the recognition motif for the nuclear transcription factor YY1. This factor regulates a multitude of cellular promoters, as an activator of transcription, as a repressor, or as an initiator element-binding protein. Antibodies directed against YY1 were used to show that YY1 copurified with the vaccinia late promoter-binding protein and was present in late promoter-protein complexes in gel supershift assays. Bacterially expressed YY1 also bound specifically to late promoter DNA. A dinucleotide replacement within the YY1 recognition motif directly adjacent to the transcription start site severely reduced the affinity of YY1 for the I1L promoter in vitro and impaired I1L promoter-dependent transcription in vivo. The intracellular localization of YY1 was shown by immunofluorescence microscopy to shift from primarily nuclear to the cytoplasm after vaccinia infection. These results indicate that YY1 has a positive role in the regulation of vaccinia virus late gene transcription and suggest that poxviruses have adapted cellular initiator elements as a means of regulating viral gene expression. This is the first identifiable cellular protein implicated in poxvirus transcription.

Vaccinia Virus Intermediate and Late Promoter Elements Are Targeted by the TATA-Binding Protein

ABSTRACT Vaccinia virus replicates in the cytoplasm of the host cell and encodes its own RNA polymerase and transcription factors. The proteins that target the poxvirus RNA polymerase to intermediate- and late-class promoters have not been identified. In this study, representatives of the intermediate and late promoters were characterized at the nucleotide level to identify essential motifs. Both intermediate and late viral promoters are shown to have an essential element suggestive of TATA boxes, which are potential targets for the TATA-binding protein (TBP). Several approaches were used to test for TBP requirement in vaccinia virus transcription, including overexpression of TBP, expression of a dominant negative mutant of TBP, RNA interference, and expression of adenovirus E1A protein, which inactivates TBP. In each case, the results support an essential role for TBP in vaccinia virus intermediate- and late-gene transcription. These findings indicate that poxviruses have integrated TBP as a central feature into an otherwise heterologous transcription system. A model for transcriptional switching, in which both intermediate and late promoter elements are targeted by TBP that recruits viral transcription factors to assemble a functional complex on their cognate promoters and a dysfunctional, repressed complex on the other class, is proposed.

Antiviral Activity of Distamycin A against Vaccinia Virus Is the Result of Inhibition of Postreplicative mRNA Synthesis

ABSTRACT Distamycin A has been described as an inhibitor of the cellular pathogenesis of vaccinia virus in culture. Distamycin is an antibiotic that specifically targets the minor groove of DNA. We show here that distamycin is a potent inhibitor of vaccinia virus replication. Pulse-labeling experiments showed that most major late proteins failed to accumulate in the presence of the antibiotic. We characterized the effect of distamycin on vaccinia virus nucleic acid biosynthesis with the goal of determining the inhibitors target. Early gene transcription was unaffected. DNA synthesis proceeded at normal rates, but DNA accumulated in large masses in the cytoplasm with no evidence of virion assembly. Transcription from the intermediate class promoter for the I1L gene was partially reduced by distamycin; however, transcription from the intermediate promoters for the three late transcription factor genes was severely inhibited. The accumulation of the late transcripts for the viral F17R and A10L genes also was severely impaired and was shown to be a direct inhibition of late promoter activity. These results indicate that inhibition of postreplicative intermediate and late transcription is the basis for inhibition of vaccinia virus by distamycin and indicate that DNA minor-groove ligands hold promise for effective anti-poxvirus drugs.

Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase.

The polypeptide encoded by the vaccinia virus open reading frame D7R was synthesized in bacteria. Immunization of rabbits with the polypeptide resulted in antibodies that specifically recognized a virion polypeptide of 20,000 daltons. The immunoreactivity with the 20,000-dalton polypeptide was found to coincide with the virion-associated DNA-dependent RNA polymerase through DEAE-cellulose chromatography and glycerol gradient sedimentation. These results argue that the product of the vaccinia open reading frame D7R is a subunit of the viral RNA polymerase.

Downregulation of vaccinia virus intermediate and late promoters by host transcription factor YY1

Approximately half of the intermediate and late gene transcriptional promoters of vaccinia virus have a binding site for the cellular transcription factor YY1 that overlaps the initiator elements. Depletion of YY1 using RNA interference enhanced the activity of these promoters, while overexpression of YY1 repressed their activity. Viral promoter nucleotide replacements that specifically impair the binding of YY1 mostly alleviated the transcriptional repression and correlated with the ability of YY1 to stably interact with the initiator DNAs in vitro. The transcriptional repression activity was localized to the C-terminal DNA-binding domain of the protein. These results indicate that YY1 functions to negatively regulate these vaccinia virus promoters by binding to their initiator elements.

Expansion of poxvirus RNA polymerase subunits sharing homology with corresponding subunits of RNA polymerase II

Poxvirus-encoded RNA polymerases were known previously to share extensive sequence homology in their two largest subunits with the corresponding subunits of cellular RNA polymerases and a modest alignment between the smallest poxvirus subunit and RBP10 of RNA polymerase II. The remaining subunits had no apparent cellular homologs. In this study, the HHpred program that combines amino acid sequence alignments with secondary structure predictions was used to search for homologs to the poxvirus RNA polymerase subunits. Significant matches of vaccinia RNA polymerase 22-, 19-, and 18-kDa subunits to RNA polymerase II subunits RPB5, 6, and 7, respectively, were identified. These results strengthen the concept that poxviral RNA polymerases likely evolved from cellular RNA polymerases.

Bidirectional transcriptional promoters in the vaccinia virus genome.

Vaccinia virus intermediate and late class transcriptional promoters each have two essential sequence elements: an initiator at the transcriptional start site and an upstream core element. Many of the transcription units in the viral genome are oriented divergently with insufficient nucleotides between the start of the open reading frames to accommodate two separate upstream core elements in their promoters. This raises the possibility that two promoters could share essential elements. Reporter gene experiments were used in this study to document examples of promoter arrangements in which two late promoters share a core element and another in which a late promoter shares a core element with an intermediate promoter. Another arrangement in which the core element of one late promoter is the initiator of the other is shown. Nucleotide replacements in the initiator element of a bidirectional promoter lead to activation of the other, suggesting that bidirectional promoter arrangement is a mechanism of attenuating promoter strength.

An In Vitro Transcription System for Studying Vaccinia Virus Early Genes

Transcription of the vaccinia virus early genes occurs within the confines of the virion core structure. Therefore, isolated virions are a particularly rich source of proteins that function in early mRNA biosynthesis. Methods are described here for the extraction of purified vaccinia virions to yield protein mixtures with high transcriptional activity on viral early gene templates, responding specifically to both transcriptional initiation and termination signals in the DNA.

The age of reverse biochemistry

In any field of science, new information comes in cycles of feast and famine. Knowledge is harvested steadily, until an impasse is encountered for lack of technological capabilities. Upon development of new methodologies, a flood of new information emerges. It appears that we are witnessing a revolution in biochemistry with new approaches in structural biology. Traditional biochemical approaches entail identification of a cellular function of interest, purification of the protein(s) responsible for the function, and characterization of its activity in vitro, culminating with solution of the atomic structure by X-ray crystallography or nuclear magnetic resonance spectroscopy. With a proliferation of structural biologists and the exponential growth in numbers of structures now available in databases, a “reverse biochemistry” approach to identification of protein function is now possible. Computational tools are now available for scanning a three-dimensional structure against databases to identify similarly structured proteins. A beautiful example of this approach is reported by Robert Liddington and colleagues in this issue of Protein Science (Aoyagi et al. 2007). Liddingtons group crystallized the vaccinia virus N1L protein, which was known to be important for viral virulence (Kotwal et al. 1989), but its function was unknown. It has no significant amino acid sequence similarity to any other protein. Comparison of the N1L structure with that of proteins in the DALI server database (Holm and Sander 1993) revealed a striking similarity to the bcl-2 family of proteins that function in modulating cellular apoptosis, or programmed cell death. A bcl-2-like function was confirmed by binding assays with peptides corresponding to interaction domains on the pro-apoptotic BH3 proteins. When a virus invades a cell, a battle ensues. A successful virus infection will result in virus multiplication, allowing the virus to spread to other cells. Higher eukaryotic cells mount several different defenses aimed at slowing or stopping the replication of the virus by the auto-destruct process of apoptosis. The cell attempts to block virus replication by dismantling its own structure (Holm and Sander 1993). The triggering of apoptosis results in inhibition of protein synthesis through a pathway regulated by the double-stranded RNA activated protein kinase, PKR. Mitochondrial function is destroyed in a process requiring the Bak and Bax proteins. Inner mitochondrial membrane potential is lost and the SMAC, TtrA2, apoptosis inducing factor, endonuclease G, and cytochrome c proteins are released from the organelle (Benedict et al. 2002), presumably by opening of the mitochondrial transition pores. The bcl-2 proteins are believed to act as signal transduction checkpoint regulators of mitochondrial destruction by modulating the activity of Bak and Bax proteins by heterodimerization (Wang 2001). The release of cytochrome c leads to the activation of caspase proteases that proceed to cleave the nuclear matrix proteins to dismantle the nucleus. Not to be outdone, viruses have the ability to counter all of these defenses. For maximal production of eukaryotic viruses, the infection generally must proceed for several days. Therefore, it is not surprising that the virus must develop some means to block apoptosis if high yields of progeny are to be produced. Another vaccinia virus protein, the F1L gene product, was previously reported to have bcl-2-like activity (Wasilenko et al. 2003). This activity was discovered through a more traditional approach of screening viruses that have genome deletions that allow increased apoptosis during infection. The vaccinia N1L protein bears six of the seven signature α-helices characteristic of bcl-2 proteins in a tight bundle. The N1L helices are marginally smaller than those of other bcl-2 proteins; however, minimized size relative to cellular counterparts is a feature common to many vaccinia virus proteins. The amino acid sequence of N1L has barely any detectable similarity to that of other family bcl-2 family members. A forced fit reveals only 11% amino acid identity with other members. Such extreme divergence of vaccinia proteins from cellular counterparts is not without precedent. The vaccinia DNA topoisomerase is structurally similar to other DNA recombinases, yet only the active site residues of the enzyme are conserved (Cheng et al. 1998). It would seem that the lesson here is that there is more than one way to build a protein.