The Ebola Virus Glycoprotein Contributes to but Is Not Sufficient for Virulence In Vivo

Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence.     

The Mitochondrial Genome of Acropora tenuis (Cnidaria; Scleractinia) Contains a Large Group I Intron and a Candidate Control Region

The complete nucleotide sequence of the mitochondrial genome of the coral Acropora tenuis has been determined. The 18,338 bp A. tenuis mitochondrial genome contains the standard metazoan complement of 13 protein-coding and two rRNA genes, but only the same two tRNA genes (trnM and trnW) as are present in the mtDNA of the sea anemone, Metridium senile. The A. tenuis nad5 gene is interrupted by a large group I intron which contains ten protein-coding genes and rns; M. senile has an intron at the same position but this contains only two protein-coding genes. Despite the large distance (about 11.5 kb) between the 5?-exon and 3?-exon boundaries, the A. tenuis nad5 gene is functional, as we were able to RT-PCR across the predicted intron splice site using total RNA from A. tenuis. As in M. senile, all of the genes in the A. tenuis mt genome have the same orientation, but their organization is completely different in these two zoantharians: The only common gene boundaries are those at each end of the group I intron and between trnM and rnl. Finally, we provide evidence that the rns-cox3 intergenic region in A. tenuis may correspond to the mitochondrial control region of higher animals. This region contains repetitive elements, and has the potential to form secondary structures of the type characteristic of vertebrate D-loops. Comparisons between a wide range of Acropora species showed that a long hairpin predicted in rns-cox3 is phylogenetically conserved, and allowed the tentative identification of conserved sequence blocks.     

Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.Influenza A virus is a member of the Orthomyxoviridae and contains a segmented, negative-sense RNA genome that codes for 10 or 11 proteins, depending upon the virus strain (11). The integral membrane protein M2 is the viral ion channel protein that is required during virus entry (29) and for the production of infectious virus particles (4, 10, 12, 13). The sequences responsible for the latter map to the cytoplasmic tail of the protein and overlap with a number of sites for posttranslational modification, which include palmitoylation and phosphorylation (7, 26, 31). Palmitoylation occurs on the cysteine present at amino acid 50 and is not required for ion channel activity of the M2 protein from A/Udorn/72 (H3N2) (7). Palmitoylation of M2 appeared to be dispensable for the production of infectious virus particles using a reassortant virus consisting of seven segments from an H3N8 subtype virus (A/Equine/Miami/63) and the M segment from an H1N1 subtype virus (A/Puerto Rico/8/34) (2). No studies examining the role of M2 palmitoylation in the context of a naturally occurring influenza A virus strain have been published to date.The significance of palmitoylation of the influenza A virus hemagglutinin (HA) protein can vary among virus strains. Palmitoylation of HA from an H7 and an H1 but not an H3 subtype is required for efficient membrane fusion (5, 24, 32), whereas palmitoylation of HA from an H3 but not an H1 subtype is required for virus assembly (5). An analysis of 3,532 sequences of influenza isolates from humans revealed that the M2 residue C50 is conserved in a strain-specific manner. A total of 2,602 of 2,610 H3N2 sequences code for a cysteine at this position; the cysteine, however, is conserved in only 330 of 1,051 H1N1 sequences (data not shown). A serine residue is substituted for cysteine in the majority of the H1N1 viruses that do not have a cytoplasmic palmitoylation site; the newly emerged 2009 H1N1 influenza A viruses, however, do have a cysteine at this position (3). The sequence alignment data are consistent with a strain-specific selective pressure to maintain the palmitoylation site on the M2 protein. Interestingly, other M2 cytoplasmic tail sequences display differential effects on infectious virus production, depending on the strain used (12).To investigate the role of M2 palmitoylation in influenza A virus replication, we substituted a serine for the cysteine residue at position 50 (C50S) of the M2 protein in two influenza A virus strains, A/Udorn/72 (H3N2) (rUdorn) and A/WSN/33 (H1N1) (rWSN). The resultant viruses were tested for their ability to replicate in tissue culture cells, and the mouse-adapted virus was tested for virulence in a mouse model of infection. Neither mutant virus showed any defect in virus replication in tissue culture cells, in differentiated murine primary trachea epithelial cells (mTEC), or in the lungs of infected mice. The viruses lacking a palmitoylation site, however, did have a modest reduction in virulence, suggesting that M2 palmitoylation is dispensable for in vitro replication but contributes to virus virulence in vivo.     

In Vivo Analysis Reveals That the Interdomain Region of the Yeast Proliferating Cell Nuclear Antigen Is Important for DNA Replication and DNA Repair

To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs(-)) and 31 methyl methanesulfonate-sensitive (Mms(s)) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mms(s) mutations, the Cs(-) mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs(-) pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs(-) pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.     

The Lysozyme-Induced Peptidoglycan N-Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.     

The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U_L) and unique short (U_S), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U_L and U_S. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U_L. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U_L, directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U_L, we concluded that our results can provide an explanation for the almost exclusive orientation of the U_L seen in packaged VZV DNA.Varicella-zoster virus ([VZV] Human Herpesvirus 3), is a highly cell-associated alphaherpesvirus that causes chicken pox (varicella) upon infection of naïve individuals (2). During primary infection, VZV is able to establish latency in cranial nerves, as well as dorsal root and autonomic ganglia, where it remains dormant until a reactivation event occurs (11). Reactivation of VZV occurs primarily in elderly or immunocompromised individuals and results in the development of shingles (herpes zoster), which is often associated with severe pain and postherpetic neuralgia (1).The VZV genome, the smallest among the human herpesviruses, is approximately 125 kbp in size and encodes at least 70 unique open reading frames (ORFs) (1). As has been reported for all alphaherpesviruses, the VZV genome consists of two unique regions, unique long (U_L) and unique short (U_S), each flanked by inverted repeat regions (TR_L, IR_L, TR_S, and IR_S) (9). In contrast to herpes simplex virus type 1 (HSV-1), the prototype alphaherpesvirus, VZV contains only very short repeats (88 bp) on either end of U_L, characteristic of members of the Varicellovirus genus (6). During alphaherpesvirus replication, four isomers of viral DNA are generated which can be distinguished by the orientation of U_L and U_S relative to each other. While all four possible isomers of HSV-1 DNA are packaged in virions as equimolar populations, virions produced by VZV and other varicelloviruses, such as equine herpesvirus type 1 (EHV-1), contain predominantly only two of the four possible isomeric forms of the genome (6, 10, 12, 15, 23). It was shown by Southern blot analysis of VZV virion DNA that inversion of the U_L region is rare and occurs in only approximately 5% of cases (6), which also may be attributed to a rare circular configuration of the genome within the virion (14). A previous report on EHV-1 suggested that inversion of the U_L region in infected cells is common but that packaging occurs in a directional manner (23). For both VZV and EHV-1, the reason for the more-or-less exclusive orientation of U_L within the virion still remains unknown.The organization of the VZV genome is similar to that of HSV-1, and over 90% of the VZV ORFs have counterparts in the HSV-1 genome (1, 13). One of the genes with a predicted HSV-1 homologue is ORFS/L, also referred to as ORF0. ORFS/L is predicted to encode a tail-anchored 157-amino-acid (aa) residue type 2 transmembrane protein and was discovered by Kemble and coworkers (13). The gene is located at the very beginning of U_L, directly adjacent to the a-like sequences that contain PacI and PacII sites crucial for the cleavage and packaging of concatameric VZV DNA (Fig. ​(Fig.1)1) (13, 20). Although no function has yet been attributed to the ORFS/L (ORF0) gene or its product, bioinformatic analysis of the VZV genome indicated that it represents a homologue of HSV-1 UL56 (RefSeq accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001348","term_id":"9625875","term_text":"NC_001348"}}NC_001348) (7, 8). While UL56 is dispensable for HSV-1 replication in vitro, it plays an important role in pathogenicity in vivo (3, 21). Little is known about the molecular mechanism of UL56 function in the case of HSV-1, but UL56 orthologues are specified by most members of the Alphaherpesvirinae subfamily (26). It was shown that the HSV-2 UL56 product localizes to the Golgi network and interacts with KIF1A, a kinesin motor protein, suggesting a role in vesicular trafficking (16, 17).Open in a separate windowFIG. 1.Overview of the VZV ORFS/L genomic region and the mutants generated. (A) Schematic representation of the VZV genome with a focus on the terminal region containing ORFS/L. Scale bars provide an accurate measure of the genome and the expanded region. (B) Overview of the mutants generated with mutations in the ORFS/L region. A cross indicates the deletion of the corresponding region. Black arrows indicate the loci of stop codon or HA tag insertion.A previous study of Kemble and coworkers also addressed the localization of the ORFS/L protein using a rabbit polyclonal antibody. It was reported that the ORFS/L product was found exclusively in the cytoplasm, which is contradictory to the findings for the HSV-2 orthologue and also to the localization of the ORFS/L protein based on in silico predictions from the primary sequence (13). ORFS/L of the P-Oka strain was recently shown to be unglycosylated but present in the virion (18). Furthermore, ORFS/L expression was detected in skin lesions of individuals, as well as neurons of dorsal root ganglia, during virus reactivation (13). In addition, the deletion of aa 29 to aa 157 of ORFS/L was shown to have an effect on viral replication in vitro and in vivo in the SCID-hu mouse model with thymus-liver implants. In this study, a virus-encoded luciferase reporter system was used to evaluate the growth properties of several bacterial artificial chromosome (BAC)-derived VZV mutants (28). However, it has remained unknown whether the observed growth defect is dependent on ORFS/L gene function or is due to the deletion of another critical sequence element.In this study, we sought to perform a systematic analysis of ORFS/L sequences. We were able to demonstrate that the ORFS/L protein localizes to the Golgi network in infected and transfected cells, providing further evidence for its predicted structure as a tail-anchored type 2 transmembrane protein and lending further support to the notion that it is the orthologue of HSV UL56. In addition, we showed that the ORFS/L gene product is important for efficient VZV replication in vitro. However, we also identified a 5′ region of the predicted ORFS/L that is essential for replication and plays a role in cleavage of viral DNA, as previously suggested by Davison and colleagues (6, 7). Since this essential region is not present at the opposite end of U_L, it could provide an explanation for the almost exclusive packaging in VZV virions of two viral DNA isomers with an invariable U_L orientation.     

The Pseudorabies Virus VP22 Homologue (UL49) Is Dispensable for Virus Growth In Vitro and Has No Effect on Virulence and Neuronal Spread in Rodents

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.     

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

Human cytomegalovirus (HCMV)-specific CD8⁺ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

CD8⁺ cytotoxic T lymphocytes (CTL) recognize virus-infected cells via the T-cell receptor (TCR), an αβ heterodimer that has specificity for the peptide antigen presented by major histocompatibility complex (MHC) class I molecules. During T-cell development in the thymus, the TCR β-chain is constructed by rearrangement of variable (V), diversity (D), and joining (J) gene segments, and the α-chain by rearrangement of V and J segments. Additional diversity is generated by imperfect joining of these segments, exonucleotide nibbling at the joins, and addition of non-germ line-encoded N-region nucleotides (25). The regions spanning the V-D-J and V-J joins constitute the hypervariable CDR3 regions which are thought to interact with the middle of the bound peptide and to account for approximately 50% of the TCR’s interaction with peptide (14, 15, 20). The α- and β-chain complementarity determining regions CDR1, which reside within the TCR V segments, are thought to interact with the N and C termini of a peptide that is bound to MHC. By contrast, Vα and Vβ CDR2s are thought to interact predominantly with the MHC itself (14, 15).Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that infects between 60 and 90% of individuals, depending on the population studied. After primary HCMV infection, the virus persists lifelong in a latent state in cells of the myeloid lineage and under the control of the immune system (5). HCMV reactivation can, however, cause serious disease in immunocompromised individuals, such as patients with advanced human immunodeficiency virus (HIV) infection (30) and patients who have undergone bone marrow transplantation (33). Evidence from animal models (32) and from studies of immunosuppressed humans (39) indicates that virus-specific CD8⁺ CTL have a role in protection against CMV disease.We previously studied in detail the HCMV-specific CTL response in healthy virus carriers. All seropositive donors had high frequencies of MHC-restricted HCMV-specific memory CTL precursors in peripheral blood and strongly recognized one of the viral tegument proteins, pp65. In some donors, the CTL response to this protein was highly focused, recognizing only a single epitope within pp65, whereas in others the CTL recognized multiple pp65 peptides (41 and unpublished data).The aim of this study was to examine the clonal composition of the memory CTL response to HCMV pp65 by determining how many different CTL clones are involved in the recognition of a given pp65 peptide. In order to do this, we analyzed the TCR α- and β-chain usage of multiple independently derived peptide-specific CTL clones from healthy virus carriers.Previous studies have examined the heterogeneity of the CTL response to other human virus infections within single subjects (2, 8, 11, 18, 19, 22, 38) or between different donors (2, 6, 8, 11, 23, 38). In the most extreme cases, a very high degree of TCR focusing has been seen: in a study of one HIV-positive individual’s CTL response to an HLA-B14-restricted HIV env peptide, the same TCR was used by 9 of 10 peptide-specific CTL clones, each derived at different time points over the course of 36 months (22). Similarly, multiple independent CTL clones specific to an HLA-B8-restricted Epstein-Barr virus (EBV) peptide derived from one virus carrier at one time point all used the same TCR (2). The CTL response to different human T-lymphotropic virus type 1 (HTLV-1) peptides has been observed to be oligoclonal within individual donors (38). However, in a variety of other human and mouse viral infections within a given individual, the repertoire of CTL specific for a given peptide has been highly heterogeneous (8, 11, 18, 19).The TCRs of CTL obtained from different donors that recognize the same peptide-MHC complex often show some conservation of gene segment usage, although they differ in hypervariable sequence. For example, Vβ segments and certain β-chain CDR3 motifs were conserved between TCR that recognized an HLA-A2-restricted influenza virus peptide in CTL clones derived from different donors (23); the same phenomenon has been seen for an HLA-B27 restricted influenza virus peptide (6) and an HLA-A11-restricted EBV peptide (8). A much higher degree of TCR conservation has also been seen; the same TCR α- and β-chain protein sequences were used by CTL clones from four of five unrelated donors that recognized an HLA-B8 restricted EBV peptide (2). In the case of HTLV-1, CTL from different donors that were specific to the same peptide used largely unrelated TCR (38).For all of the human viruses so far studied, the clonal composition of virus-specific CTL has only been examined for a very few viral peptide-MHC combinations, sometimes in only one donor or at only one time point. In this study, we have therefore examined multiple CTL clones specific to a total of four pp65 peptides, all restricted by three different HLA alleles. We have derived these clones from six healthy virus carriers at one to four time points up to 18 months apart. To identify CTL clonotypes for longitudinal studies and to determine whether HIV infection modifies the clonal composition of HCMV-specific CTL, we have also examined pp65-specific memory CTL in two asymptomatic HIV-infected subjects who are HCMV seropositive. For any given individual, whether HIV seropositive or seronegative, our results indicate that the memory CTL response to individual HCMV pp65 epitopes is highly focused and contains CTL clones that have undergone extensive expansion in vivo.     

The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose)

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.     

The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.     

The Large Subunit of Replication Factor C (Rfc1p/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces Cerevisiae

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.