Human Cytomegalovirus Reprograms the Expression of Host Micro-RNAs whose Target Networks are Required for Viral Replication: A Dissertation
AuthorsLagadinos, Alexander N.
Faculty AdvisorTimothy Kowalik, PhD
Academic ProgramImmunology and Microbiology
UMass Chan AffiliationsMicrobiology and Physiological Systems
Document TypeDoctoral Dissertation
Immunology and Infectious Disease
MetadataShow full item record
AbstractThe parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/32038
RightsCopyright is held by the author, with all rights reserved.
Showing items related by title, author, creator and subject.
Limits and patterns of cytomegalovirus genomic diversity in humansRenzette, Nicholas; Pokalyuk, Cornelia; Gibson, Laura; Bhattacharjee, Bornali; Schleiss, Mark R.; Hamprecht, Klaus; Yamamoto, Aparecida Y.; Mussi-Pinhata, Marisa M.; Britt, William J.; Jensen, Jeffrey D.; et al. (2015-07-28)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 approximately 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.
Extensive genome-wide variability of human cytomegalovirus in congenitally infected infantsRenzette, Nicholas; Bhattacharjee, Bornali; Jensen, Jeffrey D.; Gibson, Laura L.; Kowalik, Timothy F. (2011-05-19)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.
DNA vaccine prime followed by boost with live attenuated virus significantly improves antigen-specific T cell responses against human cytomegalovirusGil, Anna; Shen, Siyuan; Coley, Scott; Gibson, Laura; Diamond, Don J.; Wang, Shixia; Lu, Shan (2013-10-01)As a leading cause of congenital infection and a major threat to immunocompromised individuals, human cytomegalovirus (HCMV) is a major global public health concern. Effective HCMV vaccines would need to induce potent and balanced humoral and cellular immune responses. In this pilot study, immunogenicity studies were conducted in mice to examine HCMV antigen-specific antibody and T cell responses when a heterologous prime-boost immunization strategy was tested. DNA vaccines expressing either targets of protective antibody responses (gB and gM/gN) or well characterized T cell immunogens (pp65, pp150, and IE1) were used as the priming immunization while the live attenuated HCMV vaccine Towne strain was used as the boost, which may act like an inactivated vaccine due to the inability of HCMV to replicate in a mouse host. Our data indicate that while DNA vaccines were effective in priming HCMV-specific antibody responses, the final titers of gB- or gM-specific antibodies were not much different from those elicited by using multiple immunizations of HCMV alone. In contrast, DNA priming significantly enhanced T cell responses against gB, pp65, and IE1 as measured by IFN-gamma. However, HCMV alone was not effective in eliciting strong T cell immune responses when used in a mouse host. Our data indicate that the complexity of antigen composition from a large virus, such as HCMV, may affect the profile of immune responses when viral vaccines are used as a boost.