Philomena Ostapchuk - Academia.edu (original) (raw)

Papers by Philomena Ostapchuk

This article cites 44 articles, 18 of which can be accessed free

Journal of Virology, 1993

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and esse... more Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to ...

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI... more Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) partic...

Nucleic Acids Research, 2019

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to... more Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII– than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results ...

The EMBO Journal, 1994

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produc... more The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-termnini additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.

PLOS Pathogens, 2017

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-... more The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Molecular and Cellular Biology, 1989

Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, ... more Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, nonliver cell lines and binds to the hepatitis B virus and polyomavirus transcriptional enhancer regions in vitro. An inverted repeat (5'-GTTGCNNNGCAAC-3') is located within both binding regions. Diethyl pyrocarbonate interference binding assays and competition binding experiments using altered binding sites demonstrated that EF-C contacts symmetrical nucleotides within the inverted repeat. Mutations that changed the length of the spacer region between the arms of the inverted repeat were introduced in the hepatitis enhancer region. Introduction of 1 or 2 base pairs between the repeats did not affect EF-C binding, but deletion of 1 base pair or introduction of 3 to 9 base pairs reduced binding dramatically. Introduction of 10 base pairs restored partial EF-C binding ability. These and other results suggest that EF-C binding is stabilized by dimerization. In vivo assays for enhanc...

Nature, 2016

Viral proteins mimic host protein structure and function to redirect cellular processes and subve... more Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses 1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses 2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones 3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles 4,5 , it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified posttranslational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses 6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Current Topics in Microbiology and Immunology, 2003

ABSTRACT The application of fundamental concepts about the packaging of the adenovirus genome has... more ABSTRACT The application of fundamental concepts about the packaging of the adenovirus genome has contributed significantly to the development of therapeutic viral vectors for gene therapy. The packaging of adenovirus DNA into virus particles requires a cis-acting domain at the left end of the genome. This region contains a series of repeated sequences, termed A repeats due to their AT-rich character, that direct the packaging process. A repeats are believed to represent the binding sites for viral and cellular factors that mediate viral DNA packaging. This review will focus on fundamental aspects of adenovirus DNA packaging as well as how this information has been used and may be used to augment the selectivity of viral DNA packaging in applications pertaining to gene therapy vectors.

Virology, 1991

Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus... more Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus enhancer regions. In this paper, we have characterized new and divergent EF-C binding sites in several viral regulatory regions. We also have demonstrated that EF-C binds to certain DNA sites only when CpG dinucleotide base pairs are methylated (m5C). EF-C binds to other sites in a methylation-independent manner. Based on similar binding properties and identical binding sites, it is very likely that EF-C corresponds to the nuclear protein MDBP previously identified by virtue of binding to methylated DNA.

Proceedings of the National Academy of Sciences, 1986

We have identified a factor present in nuclear extracts of undifferentiated F9 murine embryonal c... more We have identified a factor present in nuclear extracts of undifferentiated F9 murine embryonal carcinoma cells that specifically interacts with the polyomavirus enhancer region. Nuclease "footprint" analysis was used to define the binding site that corresponds precisely to the boundaries of polyoma enhancer element C defined by Veldman et al. [Veldman, G. M., Lupton, S. & Kamen, R. (1985) Mol. Cell. Biol. 5, 649-658] that is required as an enhancer for efficient viral DNA replication and early and late region transcription. The region of nuclease protection contains a 6-base-pair inverted repeat, separated by 3 base pairs, and symmetrical flanking DNase I hypersensitive cleavage sites, suggesting that this factor may bind as a dimer. A cloned 29-base-pair polyoma DNA fragment contains an intact binding domain. Similar levels of binding activity were found in nuclear extracts prepared from differentiated murine F9 cells, as well as murine L cells and human HeLa cells. The ...

PLoS ONE, 2011

We have previously described a new family of mutant adenoviruses carrying different combinations ... more We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes.

Journal of Virology, 2011

Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immu... more Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution, thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors, including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. Here, we report a two-step labeling process involving an initial metabolic placement of a uniquely reactive unnatural amino acid, azidohomoalanine (Aha), followed by highly specific chemical modification. As genetic modification of adenovirus is unnecessary, vector production is exceedingly straightforward. Aha incorporation demonstrated no discernible impact on either virus production or infectivity of the resultant particles. “Click” chemical modification of surface-exposed azides was highly selective, allowing for the attachment of a wide range of functionality. Decoration of human ...

Journal of Virology, 2003

Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the gen... more Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis -acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans -acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding ac...

Journal of Virology, 2005

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the ge... more Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis -acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans -acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct inte...

Journal of Virology, 2003

The design of drugs for treatment of virus infections and the exploitation of viruses as drugs fo... more The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate ...

Journal of Virology, 2001

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major cha... more Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive ( ts ) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temper...

Journal of Virology, 2006

Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence o... more Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis -acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N 8 -CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral ge...

Journal of Virology, 2011

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely sim... more The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses—the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo . By utilizing a virus unable to express IVa2, pm 8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results sh...

This article cites 44 articles, 18 of which can be accessed free

Journal of Virology, 1993

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and esse... more Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to ...

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI... more Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) partic...

Nucleic Acids Research, 2019

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to... more Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII– than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results ...

The EMBO Journal, 1994

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produc... more The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-termnini additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.

PLOS Pathogens, 2017

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-... more The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein-protein VII. Two other Ad core proteins, V and X/, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Molecular and Cellular Biology, 1989

Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, ... more Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, nonliver cell lines and binds to the hepatitis B virus and polyomavirus transcriptional enhancer regions in vitro. An inverted repeat (5'-GTTGCNNNGCAAC-3') is located within both binding regions. Diethyl pyrocarbonate interference binding assays and competition binding experiments using altered binding sites demonstrated that EF-C contacts symmetrical nucleotides within the inverted repeat. Mutations that changed the length of the spacer region between the arms of the inverted repeat were introduced in the hepatitis enhancer region. Introduction of 1 or 2 base pairs between the repeats did not affect EF-C binding, but deletion of 1 base pair or introduction of 3 to 9 base pairs reduced binding dramatically. Introduction of 10 base pairs restored partial EF-C binding ability. These and other results suggest that EF-C binding is stabilized by dimerization. In vivo assays for enhanc...

Nature, 2016

Viral proteins mimic host protein structure and function to redirect cellular processes and subve... more Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses 1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses 2. Viral encoded core basic proteins compact viral genomes but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones 3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles 4,5 , it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified posttranslational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in chromatin of members of the high-mobility group protein B family (HMGB1, HMGB2, and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses 6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Current Topics in Microbiology and Immunology, 2003

ABSTRACT The application of fundamental concepts about the packaging of the adenovirus genome has... more ABSTRACT The application of fundamental concepts about the packaging of the adenovirus genome has contributed significantly to the development of therapeutic viral vectors for gene therapy. The packaging of adenovirus DNA into virus particles requires a cis-acting domain at the left end of the genome. This region contains a series of repeated sequences, termed A repeats due to their AT-rich character, that direct the packaging process. A repeats are believed to represent the binding sites for viral and cellular factors that mediate viral DNA packaging. This review will focus on fundamental aspects of adenovirus DNA packaging as well as how this information has been used and may be used to augment the selectivity of viral DNA packaging in applications pertaining to gene therapy vectors.

Virology, 1991

Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus... more Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus enhancer regions. In this paper, we have characterized new and divergent EF-C binding sites in several viral regulatory regions. We also have demonstrated that EF-C binds to certain DNA sites only when CpG dinucleotide base pairs are methylated (m5C). EF-C binds to other sites in a methylation-independent manner. Based on similar binding properties and identical binding sites, it is very likely that EF-C corresponds to the nuclear protein MDBP previously identified by virtue of binding to methylated DNA.

Proceedings of the National Academy of Sciences, 1986

We have identified a factor present in nuclear extracts of undifferentiated F9 murine embryonal c... more We have identified a factor present in nuclear extracts of undifferentiated F9 murine embryonal carcinoma cells that specifically interacts with the polyomavirus enhancer region. Nuclease "footprint" analysis was used to define the binding site that corresponds precisely to the boundaries of polyoma enhancer element C defined by Veldman et al. [Veldman, G. M., Lupton, S. & Kamen, R. (1985) Mol. Cell. Biol. 5, 649-658] that is required as an enhancer for efficient viral DNA replication and early and late region transcription. The region of nuclease protection contains a 6-base-pair inverted repeat, separated by 3 base pairs, and symmetrical flanking DNase I hypersensitive cleavage sites, suggesting that this factor may bind as a dimer. A cloned 29-base-pair polyoma DNA fragment contains an intact binding domain. Similar levels of binding activity were found in nuclear extracts prepared from differentiated murine F9 cells, as well as murine L cells and human HeLa cells. The ...

PLoS ONE, 2011

We have previously described a new family of mutant adenoviruses carrying different combinations ... more We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes.

Journal of Virology, 2011

Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immu... more Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution, thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors, including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. Here, we report a two-step labeling process involving an initial metabolic placement of a uniquely reactive unnatural amino acid, azidohomoalanine (Aha), followed by highly specific chemical modification. As genetic modification of adenovirus is unnecessary, vector production is exceedingly straightforward. Aha incorporation demonstrated no discernible impact on either virus production or infectivity of the resultant particles. “Click” chemical modification of surface-exposed azides was highly selective, allowing for the attachment of a wide range of functionality. Decoration of human ...

Journal of Virology, 2003

Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the gen... more Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis -acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans -acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding ac...

Journal of Virology, 2005

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the ge... more Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis -acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans -acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct inte...

Journal of Virology, 2003

The design of drugs for treatment of virus infections and the exploitation of viruses as drugs fo... more The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate ...

Journal of Virology, 2001

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major cha... more Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive ( ts ) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temper...

Journal of Virology, 2006

Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence o... more Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis -acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N 8 -CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral ge...

Journal of Virology, 2011

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely sim... more The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses—the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo . By utilizing a virus unable to express IVa2, pm 8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results sh...