The human cytomegalovirus (HCMV) viral chemokine gene, UL146, shows a high degree of variability in clinical isolates. The UL146-produced viral chemokine, vCXCL-1, has homology to CXC chemokines and is predicted to be an immune modulator that may contribute to the pathogenesis of HCMV infections. In the analysis of clinical isolates from congenitally infected infants, we found 11 distinct vCXCL-1 clades. Although the four cysteine residues that create two disulfide bonds providing the essential structure for CXC chemokines,are conserved, the N-loop region, which is important for receptor binding and activation, was hypervariable. One clade also contained a modified glutamic acid-leucine-arginine (ELR) motif (asparagine-glycine-arginine / NGR), which regulates binding to CXCR1 and CXCR2 receptors. Based on this sequence information, we hypothesize that these proteins differentially activate neutrophils, which may have a role in HCMV pathogenesis. To address these functional differences, we produced representative vCXCL-1 proteins from each of the 11 clades using a baculovirus protein expression system. Using competition binding assays, we have examined their binding affinities to either CXCR1 or CXCR2 expressed on HEK293 cells. All vCXCL-1s bound to CXCR2 with different binding affinities. Interestingly, only three vCXCL-1s bound to CXCR1 while the others demonstrated did not. We analyzed functional differences between the vCXCL-1s in calcium mobilization, adhesion molecule induction, and chemotaxis on human peripheral blood neutrophils (PBNs). Although the binding affinities to CXCR2 and/or CXCR1 were variable, all vCXCL-1s were capable of inducing intracellular calcium mobilization in PBNs and upregulating adhesion molecules on the surface of PBNs to similar levels as human CXCL1. However, the potency of the vCXCL-1s in the chemotaxis of neutrophils varied and was affinity independent. We also examined secondary chemokine production upon vCXCL-1 treatment on neutrophil-like HL60 T2 cells using real-time PCR. The results showed CCL22 induction was affinity dependent. Taken together, these results provide insights into the potential role of vCXCL-1 in HCMV pathogenesis and how the variability in these chemokines can affect neutrophil function.

published_or_final_version / Microbiology / Master / Master of Philosophy

Human cytomegalovirus (HCMV), a beta herpesvirus, persists indefinitely in the human host through a life-long, latent infection. HCMV is associated with life threatening pathologies in the immune naïve or compromised and, therefore, understanding of the mechanisms of viral persistence is imperative to human health. The ULb' region of the HCMV genome is selectively lost in high-passage strains of the virus, yet retained in low-passage strains. As such, the ULb' is hypothesized to play a role in immune evasion, pathogenesis, latency, and dissemination. ULb' encoded viral products are poorly characterized, hindering a mechanistic understanding of HCMV persistence. We previously defined a 3.6-kb locus spanning UL133-UL138 within the ULb' region important to viral latency. UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and translation mechanisms. We mapped the origins of each isoform through advanced bacterial artificial chromosome recombineering, where each ATG was disrupted and the resulting UL136 recombinant virus was screened for altered expression of the pUL136 isoforms. Remarkably, 8 of the 11 potential translation initiation sites encoded within the ORF are utilized to create the pUL136 isoforms. The pUL136 isoforms have distinct localization and trafficking patterns within the cell, including varying degrees of Golgi association, suggesting each isoform may interface with different cellular components and pathways. Further characterization of UL136 recombinant viruses revealed a complex, antagonistic relationship between the pUL136 isoforms. In endothelial cells, which are important to viral persistence and dissemination due to their ability to maintain a slow, "smoldering" infection, the 33- and 26-kDa isoforms promote replication, while the 25-kDa isoform enhances their combined activity, and the 23-/19-kDa isoforms repress the activity of the 25-kDa isoform. The pUL136 isoforms are also required for virus maturation in endothelial cells, where the 33-kDa is required both for virion envelopment and efficient formation of the perinuclear viral assembly compartment. In both an in vitro CD34⁺ cell culture model of latency and an in vivo NOD-scid IL2Rɣc^(null) humanized mouse model, a virus lacking the 23-/19-kDa isoforms fails to establish latency, instead replicating and disseminating with increased efficiency while viruses lacking the 33- and 26-kDa isoforms fail to efficiently reactivate or disseminate. Our data suggest that the interplay between the pUL136 isoforms maintains an intricate balance of infection that governs replication, latency, and virus dissemination, which ultimately contributes to the role of the UL133/8 locus in mediating outcomes of HCMV infection.

No description available.

Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay. The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs. A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.   The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs.

Human Cytomegalovirus (HCMV) is the most common cause of congenital infection in newborns. One mechanism for this virus to reach the fetus is to cross the placenta through the syncytiotrophoblast layer. Accumulation and protection of pathogens in the syncytiotrophoblast could affect the systemic distribution of pathogens and prolong maternal infections leading to increased incidence of fetal infections. Primary infections, reactivation or reinfection with another strain during pregnancy are risk factors for intrauterine HCMV transmission to the fetus. All lead to an active infection; however, viral load in blood or urine does not correlate with intrauterine transmission. I have shown that HCMV reversibly binds to the syncytiotrophoblast in vitro, protecting it from degradation. Furthermore, I demonstrated in vivo that HCMV is present in the placenta, even when cleared from maternal blood and urine. This evidence suggests increased potential for fetal transmission by virtue of continued virus localized at the maternal-fetal interface.

The clinical impact of human cytomegalovirus (CMV) and progression to CMV disease in immunocompromised patients has been reduced by therapeutic strategies using ganciclovir, valganciclovir, foscarnet and cidofovir. However, extensive antiviral therapy increases the risk of antiviral resistance due to mutations in the UL97 protein kinase and UL54 DNA polymerase. Co-infection with HHV-6 or HHV-7 is also associated with increased CMV reactivation and disease. Genotypic CMV antiviral resistance was identified in 38% of Australian immunocompromised patients. While UL97 mutations only were identified in 23% of patients, additional UL54 mutations, with the potential to confer multidrug resistance, were detected in 15% of patients. Antiviral resistant CMV strains were found to emerge rapidly in highly immunocompromised patients, and some strains were able to persist in the absence of selective pressure. Three new mutations were identified (UL97 - N597D, UL54 - F412S, D485N). N597D was characterised by recombinant phenotyping and conferred minimal ganciclovir resistance. Neither baculovirus nor coupled transcription/translation yielded full-length UL54 protein (pUL54; ~140 kDa) for activity assays. However, truncated pUL54 (~66 kDa) was purified after prokaryotic expression. HHV-6 and HHV-7 co-infection was a common clinical occurrence; with 36% of liver transplant recipients infected with HHV-6 (11% persistent) and 80% with HHV-7 (52% persistent). ValGCV therapy did not significantly alter the incidence of HHV-6, HHV-7 or co-infection. The most prevalent co-infection pattern was CMV, HHV-6 and HHV-7 (46%) and both CMV and HHV-7 (38%). CMV reactivation was predominantly independent of HHV-6/HHV-7, although 27% of patients had initial HHV-7 reactivation. Despite frequent co-infection, HHV-6 and HHV-7 were not associated with clinical disease, with possible exception of HHV-7 and acute cellular rejection. CMV antiviral resistance remains a significant issue in transplantation, emphasising the importance of antiviral resistance testing in an era of widespread prophylaxis. New mutations in UL97 and UL54 continue to be identified. Further characterisation of UL54 mutations using polymerase activity assays would increase our knowledge of enzymological basis of antiviral resistance. Co-infection with HHV-6 and HHV-7 is common in transplant recipients, but does not play a significant role in disease. Similar co-infection rates between valGCV-treated and untreated patients indicate that valGCV is not highly effective against HHV-6 and HHV-7.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical sciences and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed May 23, 2008. Includes bibliographical references (leaves 210-225). Online version of the print edition.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.148-174