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Timothy F Kowalik
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA

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Review
Published: 09 June 2021 in Viruses
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Human cytomegalovirus (HCMV), one of the most prevalent viruses across the globe, is a common cause of morbidity and mortality for immunocompromised individuals. Recent clinical observations have demonstrated that mixed strain infections are common and may lead to more severe disease progression. This clinical observation illustrates the complexity of the HCMV genome and emphasizes the importance of taking a population-level view of genotypic evolution. Here we review frequently sampled polymorphisms in the glycoproteins of HCMV, comparing the variable regions, and summarizing their corresponding geographic distributions observed to date. The related strain-specific immunity, including neutralization activity and antigen-specific cellular immunity, is also discussed. Given that these glycoproteins are common targets for vaccine design and anti-viral therapies, this observed genetic variation represents an important resource for future efforts to combat HCMV infections.

ACS Style

Hsuan-Yuan Wang; Sarah Valencia; Susanne Pfeifer; Jeffrey Jensen; Timothy Kowalik; Sallie Permar. Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity. Viruses 2021, 13, 1106 .

AMA Style

Hsuan-Yuan Wang, Sarah Valencia, Susanne Pfeifer, Jeffrey Jensen, Timothy Kowalik, Sallie Permar. Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity. Viruses. 2021; 13 (6):1106.

Chicago/Turabian Style

Hsuan-Yuan Wang; Sarah Valencia; Susanne Pfeifer; Jeffrey Jensen; Timothy Kowalik; Sallie Permar. 2021. "Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity." Viruses 13, no. 6: 1106.

Journal article
Published: 20 March 2019 in Proceedings of the National Academy of Sciences
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A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH–gL–UL128–UL130–UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT–mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

ACS Style

Xiaofei E; Paul Meraner; Ping Lu; Jill M. Perreira; Aaron M. Aker; William M. McDougall; Ronghua Zhuge; Gary C. Chan; Rachel M. Gerstein; Patrizia Caposio; Andrew D. Yurochko; Abraham L. Brass; Timothy F. Kowalik. OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism. Proceedings of the National Academy of Sciences 2019, 116, 7043 -7052.

AMA Style

Xiaofei E, Paul Meraner, Ping Lu, Jill M. Perreira, Aaron M. Aker, William M. McDougall, Ronghua Zhuge, Gary C. Chan, Rachel M. Gerstein, Patrizia Caposio, Andrew D. Yurochko, Abraham L. Brass, Timothy F. Kowalik. OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism. Proceedings of the National Academy of Sciences. 2019; 116 (14):7043-7052.

Chicago/Turabian Style

Xiaofei E; Paul Meraner; Ping Lu; Jill M. Perreira; Aaron M. Aker; William M. McDougall; Ronghua Zhuge; Gary C. Chan; Rachel M. Gerstein; Patrizia Caposio; Andrew D. Yurochko; Abraham L. Brass; Timothy F. Kowalik. 2019. "OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism." Proceedings of the National Academy of Sciences 116, no. 14: 7043-7052.

Review
Published: 28 January 2018 in Pathogens
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Human cytomegalovirus (HCMV) is a member of the β -herpesvirus subfamily within Herpesviridae that is nearly ubiquitous in human populations, and infection generally results only in mild symptoms. However, symptoms can be severe in immunonaive individuals, and transplacental congenital infection of HCMV can result in serious neurological sequelae. Recent work has revealed much about the demographic and selective forces shaping the evolution of congenitally transmitted HCMV both on the level of hosts and within host compartments, providing insight into the dynamics of congenital infection, reinfection, and evolution of HCMV with important implications for the development of effective treatments and vaccines.

ACS Style

Andrew M. Sackman; Susanne P. Pfeifer; Timothy F. Kowalik; Jeffrey D. Jensen. On the Demographic and Selective Forces Shaping Patterns of Human Cytomegalovirus Variation within Hosts. Pathogens 2018, 7, 16 .

AMA Style

Andrew M. Sackman, Susanne P. Pfeifer, Timothy F. Kowalik, Jeffrey D. Jensen. On the Demographic and Selective Forces Shaping Patterns of Human Cytomegalovirus Variation within Hosts. Pathogens. 2018; 7 (1):16.

Chicago/Turabian Style

Andrew M. Sackman; Susanne P. Pfeifer; Timothy F. Kowalik; Jeffrey D. Jensen. 2018. "On the Demographic and Selective Forces Shaping Patterns of Human Cytomegalovirus Variation within Hosts." Pathogens 7, no. 1: 16.

Journal article
Published: 01 March 2017 in Journal of Virology
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Intrahost and interhost assessments of viral diversity are often treated as measures of separate and distinct evolutionary processes, with numerous investigations reporting seemingly incompatible results between the two. For example, in human cytomegalovirus, the nucleotide diversity estimates are 10-fold higher for interhost data, while the number of segregating (i.e., polymorphic) sites is 6-fold lower. These results have been interpreted as demonstrating that sampled intrahost variants are strongly deleterious. In reality, however, these observations are fully consistent with standard population genetic expectations. Here, we analyze published intra- and interhost data sets within this framework, utilizing statistical inference tools to quantify the fitness effects of segregating mutations. Further, we utilize population level simulations to clarify expectations under common evolutionary models. Contrary to common claims in the literature, these results suggest that most observed polymorphisms are likely nearly neutral with regard to fitness and that standard population genetic models in fact well predict observed levels of both intra- and interhost variability. IMPORTANCE With the increasing number of evolutionary virology studies examining both intrahost and interhost patterns of genomic variation, a number of seemingly incompatible results have emerged, revolving around the far greater level of observed intrahost than interhost variation. This has led many authors to suggest that the great majority of sampled within-host polymorphisms are strongly deleterious. Here, we demonstrate that there is in fact no incompatibility of these results and, indeed, that the vast majority of sampled within-host variation is likely neutral. These results thus represent a major shift in the current view of observed viral variation.

ACS Style

Nicholas Renzette; Susanne P. Pfeifer; Sebastian Matuszewski; Timothy F. Kowalik; Jeffrey D. Jensen. On the Analysis of Intrahost and Interhost Viral Populations: Human Cytomegalovirus as a Case Study of Pitfalls and Expectations. Journal of Virology 2017, 91, 1 .

AMA Style

Nicholas Renzette, Susanne P. Pfeifer, Sebastian Matuszewski, Timothy F. Kowalik, Jeffrey D. Jensen. On the Analysis of Intrahost and Interhost Viral Populations: Human Cytomegalovirus as a Case Study of Pitfalls and Expectations. Journal of Virology. 2017; 91 (5):1.

Chicago/Turabian Style

Nicholas Renzette; Susanne P. Pfeifer; Sebastian Matuszewski; Timothy F. Kowalik; Jeffrey D. Jensen. 2017. "On the Analysis of Intrahost and Interhost Viral Populations: Human Cytomegalovirus as a Case Study of Pitfalls and Expectations." Journal of Virology 91, no. 5: 1.

Microbial local adaptation
Published: 18 December 2016 in Molecular Ecology
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Given the strong selective pressures often faced by populations when colonizing a novel habitat, the level of variation present on which selection may act is an important indicator of adaptive potential. While often discussed in an ecological context, this notion is also highly relevant in our clinical understanding of viral infection, in which the novel habitat is a new host. Thus, quantifying the factors determining levels of variation is of considerable importance for the design of improved treatment strategies. Here, we focus on such a quantification of human cytomegalovirus (HCMV) – a virus which can be transmitted across the placenta, resulting in foetal infection that can potentially cause severe disease in multiple organs. Recent studies using genomewide sequencing data have demonstrated that viral populations in some congenitally infected infants diverge rapidly over time and between tissue compartments within individuals, while in other infants, the populations remain highly stable. Here, we investigate the underlying causes of these extreme differences in observed intrahost levels of variation by estimating the underlying demographic histories of infection. Importantly, reinfection (i.e. population admixture) appears to be an important, and previously unappreciated, player. We highlight illustrative examples likely to represent a single‐population transmission from a mother during pregnancy and multiple‐population transmissions during pregnancy and after birth.

ACS Style

Cornelia Pokalyuk; Nicholas Renzette; Kristen K. Irwin; Susanne P. Pfeifer; Laura Gibson; William J. Britt; Aparecida Y. Yamamoto; Marisa M Mussi-Pinhata; Timothy F. Kowalik; Jeffrey D. Jensen. Characterizing human cytomegalovirus reinfection in congenitally infected infants: an evolutionary perspective. Molecular Ecology 2016, 26, 1980 -1990.

AMA Style

Cornelia Pokalyuk, Nicholas Renzette, Kristen K. Irwin, Susanne P. Pfeifer, Laura Gibson, William J. Britt, Aparecida Y. Yamamoto, Marisa M Mussi-Pinhata, Timothy F. Kowalik, Jeffrey D. Jensen. Characterizing human cytomegalovirus reinfection in congenitally infected infants: an evolutionary perspective. Molecular Ecology. 2016; 26 (7):1980-1990.

Chicago/Turabian Style

Cornelia Pokalyuk; Nicholas Renzette; Kristen K. Irwin; Susanne P. Pfeifer; Laura Gibson; William J. Britt; Aparecida Y. Yamamoto; Marisa M Mussi-Pinhata; Timothy F. Kowalik; Jeffrey D. Jensen. 2016. "Characterizing human cytomegalovirus reinfection in congenitally infected infants: an evolutionary perspective." Molecular Ecology 26, no. 7: 1980-1990.

Journal article
Published: 06 July 2015 in Proceedings of the National Academy of Sciences
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Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with ∼25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts.

ACS Style

Nicholas Renzette; Cornelia Pokalyuk; Laura Gibson; Bornali Bhattacharjee; Mark R. Schleiss; Klaus Hamprecht; Aparecida Y. Yamamoto; Marisa M Mussi-Pinhata; William J. Britt; Jeffrey D. Jensen; Timothy F. Kowalik. Limits and patterns of cytomegalovirus genomic diversity in humans. Proceedings of the National Academy of Sciences 2015, 112, E4120 -E4128.

AMA Style

Nicholas Renzette, Cornelia Pokalyuk, Laura Gibson, Bornali Bhattacharjee, Mark R. Schleiss, Klaus Hamprecht, Aparecida Y. Yamamoto, Marisa M Mussi-Pinhata, William J. Britt, Jeffrey D. Jensen, Timothy F. Kowalik. Limits and patterns of cytomegalovirus genomic diversity in humans. Proceedings of the National Academy of Sciences. 2015; 112 (30):E4120-E4128.

Chicago/Turabian Style

Nicholas Renzette; Cornelia Pokalyuk; Laura Gibson; Bornali Bhattacharjee; Mark R. Schleiss; Klaus Hamprecht; Aparecida Y. Yamamoto; Marisa M Mussi-Pinhata; William J. Britt; Jeffrey D. Jensen; Timothy F. Kowalik. 2015. "Limits and patterns of cytomegalovirus genomic diversity in humans." Proceedings of the National Academy of Sciences 112, no. 30: E4120-E4128.

Preprint content
Published: 25 September 2014
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Human cytomegalovirus (HCMV) is exquisitely adapted to the human host, and much research has focused on its evolution over long timescales spanning millennia. Here, we review recent data exploring the evolution of the virus on much shorter timescales, on the order of days or months. We describe the intrahost genetic diversity of the virus isolated from humans, and how this diversity contributes to HCMV spatiotemporal evolution. We propose mechanisms to explain the high levels of intrahost diversity and discuss how this new information may shed light on HCMV infection and pathogenesis.

ACS Style

Nicholas Renzette; Laura Gibson; Jeffrey D. Jensen; Timothy F. Kowalik. Human Cytomegalovirus Intrahost Evolution--A New Avenue for Understanding and Controlling Herpesvirus Infections. 2014, 009571 .

AMA Style

Nicholas Renzette, Laura Gibson, Jeffrey D. Jensen, Timothy F. Kowalik. Human Cytomegalovirus Intrahost Evolution--A New Avenue for Understanding and Controlling Herpesvirus Infections. . 2014; ():009571.

Chicago/Turabian Style

Nicholas Renzette; Laura Gibson; Jeffrey D. Jensen; Timothy F. Kowalik. 2014. "Human Cytomegalovirus Intrahost Evolution--A New Avenue for Understanding and Controlling Herpesvirus Infections." , no. : 009571.

Review article
Published: 25 August 2014 in Current Opinion in Virology
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Human cytomegalovirus (HCMV) is exquisitely adapted to the human host, and much research has focused on its evolution over long timescales spanning millennia. Here, we review recent data exploring the evolution of the virus on much shorter timescales, on the order of days or months. We describe the intrahost genetic diversity of the virus isolated from humans, and how this diversity contributes to HCMV spatiotemporal evolution. We propose mechanisms to explain the high levels of intrahost diversity and discuss how this new information may shed light on HCMV infection and pathogenesis.

ACS Style

Nicholas Renzette; Laura Gibson; Jeffrey D Jensen; Timothy F Kowalik. Human cytomegalovirus intrahost evolution—a new avenue for understanding and controlling herpesvirus infections. Current Opinion in Virology 2014, 8, 109 -115.

AMA Style

Nicholas Renzette, Laura Gibson, Jeffrey D Jensen, Timothy F Kowalik. Human cytomegalovirus intrahost evolution—a new avenue for understanding and controlling herpesvirus infections. Current Opinion in Virology. 2014; 8 ():109-115.

Chicago/Turabian Style

Nicholas Renzette; Laura Gibson; Jeffrey D Jensen; Timothy F Kowalik. 2014. "Human cytomegalovirus intrahost evolution—a new avenue for understanding and controlling herpesvirus infections." Current Opinion in Virology 8, no. : 109-115.

Review
Published: 23 May 2014 in Viruses
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Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed.

ACS Style

Xiaofei E; Timothy F. Kowalik. The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses. Viruses 2014, 6, 2155 -2185.

AMA Style

Xiaofei E, Timothy F. Kowalik. The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses. Viruses. 2014; 6 (5):2155-2185.

Chicago/Turabian Style

Xiaofei E; Timothy F. Kowalik. 2014. "The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses." Viruses 6, no. 5: 2155-2185.

Journal article
Published: 11 December 2013 in Journal of Virology
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Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.

ACS Style

Xiaofei E; George Savidis; Christopher Chin; Shixia Wang; Shan Lu; Abraham L. Brass; Timothy F. Kowalik. A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication. Journal of Virology 2013, 88, 2279 -2290.

AMA Style

Xiaofei E, George Savidis, Christopher Chin, Shixia Wang, Shan Lu, Abraham L. Brass, Timothy F. Kowalik. A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication. Journal of Virology. 2013; 88 (4):2279-2290.

Chicago/Turabian Style

Xiaofei E; George Savidis; Christopher Chin; Shixia Wang; Shan Lu; Abraham L. Brass; Timothy F. Kowalik. 2013. "A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication." Journal of Virology 88, no. 4: 2279-2290.

Research article
Published: 26 September 2013 in PLoS Genetics
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Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ∼88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13–18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥34 of the 167 open reading frames (∼20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization. The large, dsDNA virus Human cytomegalovirus (HCMV) is the most genetically complex viral pathogen of humans. HCMV populations are highly variable, which may allow the virus to evolve in human hosts on short timescales. We tested this hypothesis by longitudinally sampling HCMV populations from the urine and/or plasma of congenitally infected infants. We found that HCMV is generally stable within a compartment, but rapidly evolves when crossing host compartments. In fact, HCMV sampled from two compartments of the same host is as different as HCMV collected from unrelated hosts. We used mathematical modeling and population genetic analysis to show that both a bottleneck (i.e., a reduction in population size) associated with compartment colonization as well as positive selection are necessary to explain the observed differences between compartments. We also conclude from these data that fetal infection in these patients occurred between 13–18 weeks gestational age, consistent with the timing of symptomatic congenital HCMV infections. This study is the most detailed investigation of DNA virus evolution in human hosts to date, provides a framework for the study of other viral infections using similar techniques, and will aid in the development of new antiviral therapies and vaccines.

ACS Style

Nicholas Renzette; Laura Gibson; Bornali Bhattacharjee; Donna Fisher; Mark R. Schleiss; Jeffrey D. Jensen; Timothy F. Kowalik. Rapid Intrahost Evolution of Human Cytomegalovirus Is Shaped by Demography and Positive Selection. PLoS Genetics 2013, 9, e1003735 .

AMA Style

Nicholas Renzette, Laura Gibson, Bornali Bhattacharjee, Donna Fisher, Mark R. Schleiss, Jeffrey D. Jensen, Timothy F. Kowalik. Rapid Intrahost Evolution of Human Cytomegalovirus Is Shaped by Demography and Positive Selection. PLoS Genetics. 2013; 9 (9):e1003735.

Chicago/Turabian Style

Nicholas Renzette; Laura Gibson; Bornali Bhattacharjee; Donna Fisher; Mark R. Schleiss; Jeffrey D. Jensen; Timothy F. Kowalik. 2013. "Rapid Intrahost Evolution of Human Cytomegalovirus Is Shaped by Demography and Positive Selection." PLoS Genetics 9, no. 9: e1003735.

Journal article
Published: 15 June 2012 in Journal of Virology
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Human cytomegalovirus (HCMV) has been found in malignant gliomas at variable frequencies with efforts to date focused on characterizing the role(s) of single gene products in disease. Here, we reexamined the HCMV prevalence in malignant gliomas using different methods and began to dissect the genetics of HCMV in tumors. HCMV DNA was found in 16/17 (94%) tumor specimens. Viral DNA copy numbers were found to be low and variable, ranging from 10 2 to 10 6 copies/500 ng of total DNA. The tumor tissues had incongruences between viral DNA copy numbers and protein levels. However, nonlatent protein expression was detected in many tumors. The viral UL83 gene, encoding pp65, was found to segregate into five cancer-associated genotypes with a bias for amino acid changes in glioblastoma multiforme (GBM) in comparison to the low-grade tumors. Deep sequencing of a GBM-associated viral population resulted in 81,224 bp of genome coverage. Sequence analysis revealed the presence of intact open reading frames and higher numbers of high-frequency variations within the repeat long region compared to the unique long region, which harbors many core genes, and the unique short region ( P = 0.001). This observation was in congruence with phylogenetic analyses across replication-competent viral strains in databases. The tumor-associated viral population was less variable (π = 0.1% and π AA = 0.08%) than that observed in other clinical infections. Moreover, 42/46 (91.3%) viral genes analyzed had dN/dS scores of <1, which is indicative of high amino acid sequence conservation. Taken together, these findings raise the possibility that replication-competent HCMV may exist in malignant gliomas.

ACS Style

Bornali Bhattacharjee; Nicholas Renzette; Timothy F. Kowalik. Genetic Analysis of Cytomegalovirus in Malignant Gliomas. Journal of Virology 2012, 86, 6815 -6824.

AMA Style

Bornali Bhattacharjee, Nicholas Renzette, Timothy F. Kowalik. Genetic Analysis of Cytomegalovirus in Malignant Gliomas. Journal of Virology. 2012; 86 (12):6815-6824.

Chicago/Turabian Style

Bornali Bhattacharjee; Nicholas Renzette; Timothy F. Kowalik. 2012. "Genetic Analysis of Cytomegalovirus in Malignant Gliomas." Journal of Virology 86, no. 12: 6815-6824.

Journal article
Published: 21 March 2012 in Journal of Virology
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Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.

ACS Style

Xiaofei E; Bradford M. Stadler; Michelle Debatis; Shixia Wang; Shan Lu; Timothy F. Kowalik. RNA Interference-Mediated Targeting of Human Cytomegalovirus Immediate-Early or Early Gene Products Inhibits Viral Replication with Differential Effects on Cellular Functions. Journal of Virology 2012, 86, 5660 -5673.

AMA Style

Xiaofei E, Bradford M. Stadler, Michelle Debatis, Shixia Wang, Shan Lu, Timothy F. Kowalik. RNA Interference-Mediated Targeting of Human Cytomegalovirus Immediate-Early or Early Gene Products Inhibits Viral Replication with Differential Effects on Cellular Functions. Journal of Virology. 2012; 86 (10):5660-5673.

Chicago/Turabian Style

Xiaofei E; Bradford M. Stadler; Michelle Debatis; Shixia Wang; Shan Lu; Timothy F. Kowalik. 2012. "RNA Interference-Mediated Targeting of Human Cytomegalovirus Immediate-Early or Early Gene Products Inhibits Viral Replication with Differential Effects on Cellular Functions." Journal of Virology 86, no. 10: 5660-5673.

Research article
Published: 19 May 2011 in PLOS Pathogens
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Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus. Human Cytomegalovirus (HCMV) is a dsDNA virus that is the leading source of birth defects associated with an infectious agent. There is currently no effective HCMV vaccine and few treatment strategies for congenital infections exist. Thus, a better understanding of HCMV infections is warranted. Limited data has shown that HCMV exists as a mixture of a few genotypes in human hosts. Here, we describe our use of high throughput sequencing to study the extent of genome wide variability within HCMV infections sampled from congenital infections. Surprisingly, we find that HCMV populations are as variable as quasispecies RNA viruses; it is commonly believed that DNA viruses are more genetically stable than RNA viruses, and thus produce homogenous populations. Additionally, we find evidence of evolutionary pressures acting on the HCMV genome, both within and among populations. These results provide the first evidence that diversity of DNA virus populations can be comparable to that of RNA virus populations.

ACS Style

Nicholas Renzette; Bornali Bhattacharjee; Jeffrey Jensen; Laura Gibson; Timothy F. Kowalik. Extensive Genome-Wide Variability of Human Cytomegalovirus in Congenitally Infected Infants. PLOS Pathogens 2011, 7, e1001344 .

AMA Style

Nicholas Renzette, Bornali Bhattacharjee, Jeffrey Jensen, Laura Gibson, Timothy F. Kowalik. Extensive Genome-Wide Variability of Human Cytomegalovirus in Congenitally Infected Infants. PLOS Pathogens. 2011; 7 (5):e1001344.

Chicago/Turabian Style

Nicholas Renzette; Bornali Bhattacharjee; Jeffrey Jensen; Laura Gibson; Timothy F. Kowalik. 2011. "Extensive Genome-Wide Variability of Human Cytomegalovirus in Congenitally Infected Infants." PLOS Pathogens 7, no. 5: e1001344.

Research article
Published: 12 May 2011 in PLOS Pathogens
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DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses. As intracellular parasites, viruses often redirect cellular pathways to facilitate their own replication. Infection by DNA viruses often lead to the activation of host DNA damage response pathways, which normally function to repair damage to host chromosomes. Some DNA viruses depend on this infection-induced DNA damage response to efficiently replicate. How infection activates the DNA damage response is poorly understood. To address this limitation, we first determined whether the DNA damage response affects the replication of human cytomegalovirus (HCMV) and then addressed how infection induces this response in cells. We find that HCMV infection results in a host DNA damage response centered on the Ataxia Telangiectasia Mutated (ATM) protein kinase. We also show that HCMV requires ATM for efficient replication. Unexpectedly, we find that the mechanism responsible for ATM activation is the expression of E2F1, a cellular transcription factor. Moreover, expression of E2F1, like ATM, is required for HCMV replication. These observations may be of fundamental importance because infection by most DNA viruses result in both E2F1 expression and an ATM-mediated DNA damage response.

ACS Style

Xiaofei E; Mary Pickering; Michelle Debatis; Jonathan Castillo; Alexander Lagadinos; Shixia Wang; Shan Lu; Timothy F. Kowalik. An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus. PLOS Pathogens 2011, 7, e1001342 .

AMA Style

Xiaofei E, Mary Pickering, Michelle Debatis, Jonathan Castillo, Alexander Lagadinos, Shixia Wang, Shan Lu, Timothy F. Kowalik. An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus. PLOS Pathogens. 2011; 7 (5):e1001342.

Chicago/Turabian Style

Xiaofei E; Mary Pickering; Michelle Debatis; Jonathan Castillo; Alexander Lagadinos; Shixia Wang; Shan Lu; Timothy F. Kowalik. 2011. "An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus." PLOS Pathogens 7, no. 5: e1001342.

Journal article
Published: 06 October 2008 in Oncogene
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The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

ACS Style

Mary Pickering; B M Stadler; T F Kowalik. miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression. Oncogene 2008, 28, 140 -145.

AMA Style

Mary Pickering, B M Stadler, T F Kowalik. miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression. Oncogene. 2008; 28 (1):140-145.

Chicago/Turabian Style

Mary Pickering; B M Stadler; T F Kowalik. 2008. "miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression." Oncogene 28, no. 1: 140-145.

Journal article
Published: 23 January 2006 in Oncogene
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Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes.

ACS Style

F M Frame; H A Rogoff; M T Pickering; W D Cress; T F Kowalik. E2F1 induces MRN foci formation and a cell cycle checkpoint response in human fibroblasts. Oncogene 2006, 25, 3258 -3266.

AMA Style

F M Frame, H A Rogoff, M T Pickering, W D Cress, T F Kowalik. E2F1 induces MRN foci formation and a cell cycle checkpoint response in human fibroblasts. Oncogene. 2006; 25 (23):3258-3266.

Chicago/Turabian Style

F M Frame; H A Rogoff; M T Pickering; W D Cress; T F Kowalik. 2006. "E2F1 induces MRN foci formation and a cell cycle checkpoint response in human fibroblasts." Oncogene 25, no. 23: 3258-3266.

Journal article
Published: 26 September 2005 in Oncogene
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Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes.

ACS Style

Mary Pickering; T F Kowalik. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene 2005, 25, 746 -755.

AMA Style

Mary Pickering, T F Kowalik. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005; 25 (5):746-755.

Chicago/Turabian Style

Mary Pickering; T F Kowalik. 2005. "Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation." Oncogene 25, no. 5: 746-755.

Journal article
Published: 01 September 2005 in Journal of Virology
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Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type ( p53 +/+ ) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19 Arf and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G 1 to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

ACS Style

Jonathan P. Castillo; Fiona M. Frame; Harry A. Rogoff; Mary T. Pickering; Andrew D. Yurochko; Timothy F. Kowalik. Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response. Journal of Virology 2005, 79, 11467 -11475.

AMA Style

Jonathan P. Castillo, Fiona M. Frame, Harry A. Rogoff, Mary T. Pickering, Andrew D. Yurochko, Timothy F. Kowalik. Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response. Journal of Virology. 2005; 79 (17):11467-11475.

Chicago/Turabian Style

Jonathan P. Castillo; Fiona M. Frame; Harry A. Rogoff; Mary T. Pickering; Andrew D. Yurochko; Timothy F. Kowalik. 2005. "Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response." Journal of Virology 79, no. 17: 11467-11475.

Journal article
Published: 01 July 2004 in Cell Cycle
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Proper regulation of cellular proliferation is critical for normal development andcancer prevention. Most, if not all, cancer cells contain mutations in the Rb/E2F pathway,which controls cellular proliferation. Inactivation of the retinoblastoma (Rb) family ofproteins can occur through Rb loss, mutation, or inactivation by cellular or viraloncoproteins leading to unrestrained proliferation and, often times, results in apoptosis.The loss of growth control occurs primarily by derepression and activation of the E2Ftranscription factors. E2F1 in particular, serves as the primary link between loss of Rbfunction and activation of p53-dependent apoptosis. E2F1 function is crucial forresponding to loss of proper Rb-mediated growth control to activate p53 and theapoptotic program. Recently, we described the requirement for the DNA damageresponse proteins Atm, Nbs1, and Chk2 in the E2F1 apoptosis pathway. These findingssuggest that there may be a more intimate relationship between the apoptosis pathwaysresulting from loss of proper Rb-mediated growth control and apoptosis resulting fromthe accumulation of DNA damage.

ACS Style

Harry A. Rogoff; Timothy F. Kowalik. Life, Death, and E2F: Linking Proliferation Control and DNA Damage Signaling via E2F1. Cell Cycle 2004, 3, 843 -844.

AMA Style

Harry A. Rogoff, Timothy F. Kowalik. Life, Death, and E2F: Linking Proliferation Control and DNA Damage Signaling via E2F1. Cell Cycle. 2004; 3 (7):843-844.

Chicago/Turabian Style

Harry A. Rogoff; Timothy F. Kowalik. 2004. "Life, Death, and E2F: Linking Proliferation Control and DNA Damage Signaling via E2F1." Cell Cycle 3, no. 7: 843-844.