Kristine Yoder - Academia.edu (original) (raw)
Papers by Kristine Yoder
Biomolecules, 2021
Integrases of different retroviruses assemble as functional complexes with varying multimers of t... more Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data high...
This article cites 64 articles, 20 of which can be accessed free
Journal of Virology, 1997
The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role i... more The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human os...
Journal of Virology, 2016
The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance nece... more The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Rec...
The EMBO Journal, 2001
Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA... more Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to puri®ed retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal.
Proceedings of the National Academy of Sciences, 2003
Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesti... more Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesting that this region may harbor a tumor suppressor gene. By constructing a physical map of the LOH11CR2 minimal region of loss on 11q23-q24 associated with lung and breast carcinomas, we were able to clone a hereditary translocation, t(11;12)(q23;q24), in a patient with early-onset breast cancer and family history of cancer. The breakpoint was found within 6 kb of the BCSC-1 candidate tumor suppressor gene located in the LOH11CR2 region whereas additional loss of heterozygosity (LOH) analysis in breast and ovarian tumors, including that of the patient with the t(11;12)(q23;q24), implicated the BCSC-1 locus as the primary target of deletion. Northern analysis of the BCSC-1 mRNA revealed a lack of expression in 33 of 41 (80%) tumor cell lines, and its ectopic expression led to the suppression of colony formation in vitro and tumorigenicity in vivo . These data suggest that BCSC-1 may exert ...
Proceedings of the National Academy of Sciences, 2006
Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A... more Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in ...
Molecular and Cellular Biology, 2007
Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpo... more Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulat...
Journal of Virology, 2000
To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that... more To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by st...
Journal of Virology, 2000
Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Pr... more Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuc...
Clinical Cancer Research, 2004
Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the co... more Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the common fragile site FRA6E on human chromosome 6q25-q27, is associated with a frequent loss of heterozygosity and altered expression in breast and ovarian carcinomas. In addition, homozygous deletions of exon 2 creating deleterious truncations of the Parkin transcript were observed in the lung adenocarcinoma cell lines Calu-3 and H-1573, suggesting that the loss of this locus and the resulting changes in its expression are involved in the development of these tumors. Experimental Design: We examined 20 paired normal and non-small cell lung cancer samples for the presence of Parkin alterations in the coding sequence and changes in gene expression. We also restored gene expression in the Parkin-deficient lung carcinoma cell line H460 by use of a recombinant lentivirus containing the wild-type Parkin cDNA. Results: Loss of heterozygosity analysis identified a common region of loss in the Parki...
American Journal of Respiratory and Critical Care Medicine, 2005
In addition to Kaposi&amp... more In addition to Kaposi's sarcoma, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) has been associated with two other diseases: primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. Recently, evidence of KSHV infection was reported in plexiform lesions of idiopathic pulmonary arterial hypertension (IPAH) as well as in adjacent parenchyma and bronchial epithelial cells. To further investigate a possible association of KSHV and pulmonary arterial hypertension. Twenty-six lungs explanted from German patients suffering from IPAH were tested for the presence of KSHV antigen and genomes by immunohistochemistry (IHC) and polymerase chain reaction (PCR). When stained with a commercial monoclonal antibody directed against the latency-associated nuclear antigen of KSHV, LANA-1, a positive signal reminiscent of the "speckled" nuclear pattern typical of latently KSHV-infected cells was found in 16 (61.5%) cases. Alveolar and bronchial epithelial cells in areas of unremarkable lung tissue, but not cells within the plexiform lesions, were the predominantly stained cell types. Different KSHV-PCR assays (based on orf26, orfK6, and orf72) performed on samples that had tested positively in IHC, however, could not confirm KSHV infection, indicating that the IHC signal was not due to KSHV infection. One IHC-negative patient tested positive by PCR. A PCR based on consensus degenerate hybrid oligonucleotide primers (CODEHOP) to detect yet unknown gamma-herpesviruses did not reveal any specific sequences. KSHV is unlikely to play a role in the pathogenesis of IPAH.
PLoS ONE, 2014
Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. Thes... more Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polb). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polb null cell lines complemented with active site point mutants of Polb. A DNA synthesis defective mutant, but not a 59dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polb DNA synthesis activity is not necessary while 59dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.
Frontiers in Cellular and Infection Microbiology, 2020
CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukem... more CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4+ T lymphocytes that survive for the lifetime of the host. The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-...
Frontiers in Molecular Biosciences
Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome ... more Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for ...
Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleo... more Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the virus-encoded integrase (IN) and the reverse transcribed (RT) virus copy DNA (cDNA). Previous studies suggested that integration was limited by intasome-host DNA recognition progressions. Using single molecule Forster resonance energy transfer (smFRET) we show that PFV intasomes pause at nicked and gapped DNA, which targeted site-directed integration without inducing significant intasome conformational alterations. Base excision repair (BER) components that affect retroviral integration in vivo produce similar nick/gap intermediates during DNA lesion processing. Intasome pause dynamics was modified by the 5′-nick-gap chemistry, while an 8-oxo-guanine lesion, a mismatch, or a nucleotide insertion that induce backbone flexibility and/or static bends had no effect. These results suggest that dynamic often non-productive intasome-DNA interactions may...
Integrase enzymes of different retroviruses assemble as functional complexes with varying multime... more Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these da...
Biomolecules, 2021
Integrases of different retroviruses assemble as functional complexes with varying multimers of t... more Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data high...
This article cites 64 articles, 20 of which can be accessed free
Journal of Virology, 1997
The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role i... more The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human os...
Journal of Virology, 2016
The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance nece... more The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Rec...
The EMBO Journal, 2001
Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA... more Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation. NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to puri®ed retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal.
Proceedings of the National Academy of Sciences, 2003
Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesti... more Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesting that this region may harbor a tumor suppressor gene. By constructing a physical map of the LOH11CR2 minimal region of loss on 11q23-q24 associated with lung and breast carcinomas, we were able to clone a hereditary translocation, t(11;12)(q23;q24), in a patient with early-onset breast cancer and family history of cancer. The breakpoint was found within 6 kb of the BCSC-1 candidate tumor suppressor gene located in the LOH11CR2 region whereas additional loss of heterozygosity (LOH) analysis in breast and ovarian tumors, including that of the patient with the t(11;12)(q23;q24), implicated the BCSC-1 locus as the primary target of deletion. Northern analysis of the BCSC-1 mRNA revealed a lack of expression in 33 of 41 (80%) tumor cell lines, and its ectopic expression led to the suppression of colony formation in vitro and tumorigenicity in vivo . These data suggest that BCSC-1 may exert ...
Proceedings of the National Academy of Sciences, 2006
Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A... more Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in ...
Molecular and Cellular Biology, 2007
Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpo... more Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulat...
Journal of Virology, 2000
To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that... more To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by st...
Journal of Virology, 2000
Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Pr... more Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuc...
Clinical Cancer Research, 2004
Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the co... more Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the common fragile site FRA6E on human chromosome 6q25-q27, is associated with a frequent loss of heterozygosity and altered expression in breast and ovarian carcinomas. In addition, homozygous deletions of exon 2 creating deleterious truncations of the Parkin transcript were observed in the lung adenocarcinoma cell lines Calu-3 and H-1573, suggesting that the loss of this locus and the resulting changes in its expression are involved in the development of these tumors. Experimental Design: We examined 20 paired normal and non-small cell lung cancer samples for the presence of Parkin alterations in the coding sequence and changes in gene expression. We also restored gene expression in the Parkin-deficient lung carcinoma cell line H460 by use of a recombinant lentivirus containing the wild-type Parkin cDNA. Results: Loss of heterozygosity analysis identified a common region of loss in the Parki...
American Journal of Respiratory and Critical Care Medicine, 2005
In addition to Kaposi&amp... more In addition to Kaposi's sarcoma, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) has been associated with two other diseases: primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. Recently, evidence of KSHV infection was reported in plexiform lesions of idiopathic pulmonary arterial hypertension (IPAH) as well as in adjacent parenchyma and bronchial epithelial cells. To further investigate a possible association of KSHV and pulmonary arterial hypertension. Twenty-six lungs explanted from German patients suffering from IPAH were tested for the presence of KSHV antigen and genomes by immunohistochemistry (IHC) and polymerase chain reaction (PCR). When stained with a commercial monoclonal antibody directed against the latency-associated nuclear antigen of KSHV, LANA-1, a positive signal reminiscent of the "speckled" nuclear pattern typical of latently KSHV-infected cells was found in 16 (61.5%) cases. Alveolar and bronchial epithelial cells in areas of unremarkable lung tissue, but not cells within the plexiform lesions, were the predominantly stained cell types. Different KSHV-PCR assays (based on orf26, orfK6, and orf72) performed on samples that had tested positively in IHC, however, could not confirm KSHV infection, indicating that the IHC signal was not due to KSHV infection. One IHC-negative patient tested positive by PCR. A PCR based on consensus degenerate hybrid oligonucleotide primers (CODEHOP) to detect yet unknown gamma-herpesviruses did not reveal any specific sequences. KSHV is unlikely to play a role in the pathogenesis of IPAH.
PLoS ONE, 2014
Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. Thes... more Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polb). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polb null cell lines complemented with active site point mutants of Polb. A DNA synthesis defective mutant, but not a 59dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polb DNA synthesis activity is not necessary while 59dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.
Frontiers in Cellular and Infection Microbiology, 2020
CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukem... more CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4+ T lymphocytes that survive for the lifetime of the host. The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-...
Frontiers in Molecular Biosciences
Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome ... more Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for ...
Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleo... more Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the virus-encoded integrase (IN) and the reverse transcribed (RT) virus copy DNA (cDNA). Previous studies suggested that integration was limited by intasome-host DNA recognition progressions. Using single molecule Forster resonance energy transfer (smFRET) we show that PFV intasomes pause at nicked and gapped DNA, which targeted site-directed integration without inducing significant intasome conformational alterations. Base excision repair (BER) components that affect retroviral integration in vivo produce similar nick/gap intermediates during DNA lesion processing. Intasome pause dynamics was modified by the 5′-nick-gap chemistry, while an 8-oxo-guanine lesion, a mismatch, or a nucleotide insertion that induce backbone flexibility and/or static bends had no effect. These results suggest that dynamic often non-productive intasome-DNA interactions may...
Integrase enzymes of different retroviruses assemble as functional complexes with varying multime... more Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these da...