Discovery and Characterization of Cytomegalovirus Inhibitors using Reporter-based Antiviral Assays

ABSTRACT DISCOVERY AND CHARACTERIZATION OF CYTOMEGALOVIRUS INHIBITORS USING REPORTER-BASED ANTIVIRAL ASSAYS By Amine Ourahmane, MS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, August 2017 Major Director: Michael McVoy, Ph.D. Professor, Department of Pediatrics and Microbiology and Immunology Human cytomegalovirus (HCMV), a member of the herpesvirus family, causes significant disease in immunocompromised patients and is the major infectious cause of birth defects when acquired congenitally. Current HCMV antivirals are suboptimal due to modest potency, significant toxicities, and emergence of resistance. Because HCMV does not infect non-human species, related animal cytomegaloviruses are used as animal models. Of the small animal cytomegaloviruses only guinea pig cytomegalovirus (GPCMV) has been found to cross the placenta to cause fetal infection and disease. Thus, the GPCMV/guinea pig model of congenital infection can be used to study the effectiveness of vaccines or small molecule inhibitors in preventing or treating congenital infections. However, not all antivirals that inhibit HCMV are active against GPCMV. In Aim 1 of the current studies a luciferase-based assay was developed and used to determine the sensitivity of GPCMV to three novel inhibitory compounds, BDCRB, BAY 38-4766, and letermovir, which block DNA maturation of HCMV by targeting the viral terminase complex. BDCRB and BAY 38-4766 were active against GPCMV.Unfortunately, letermovir, which recently completed phase 3 clinical testing, was not active against GPCMV at concentrations up to 100 mM. In Aim 2 the mechanism of action of BDCRB against GPCMV was explored by characterizing an L406P mutation in the GP89 terminase subunit that had been previously identified in a BDCRB-resistant GPCMV. In silico homology modeling was used to identify the location of the L406P mutation in a predicted 3-D structure of GP89. That it was not located near a putative BDCRB-binding pocket (which was predicted based on confirmed resistance mutations in the homologous HCMV UL89 subunit) suggested that L406P may not confer BDCRB resistance in GPCMV. That L406P does not confer BDCRB resistance was confirmed by genetic transfer of the L406 mutation into an otherwise wild type GPCMV background and demonstration, using the luciferase-based assay, that the IC50 of BDCRB was not significantly altered (i.e., the virus containing the L406 mutation was not resistant to BDCRB). In Aim 3 a green fluorescent protein-based assay was used to evaluate four candidate compounds for antiviral activity against HCMV. These highly positively charged compounds, TriplatinNC, DiplatinNC, [Pt(dien)(Xan)]2+ and Werner’s Complex, were hypothesized to interfere with viral binding to cell surface glycosaminoglycans and thereby interfere with viral attachment and subsequent entry

LoReTTA, a user-friendly tool for assembling viral genomes from PacBio sequence data

Long-read, single-molecule DNA sequencing technologies have triggered a revolution in genomics by enabling the determination of large, reference-quality genomes in ways that overcome some of the limitations of short-read sequencing. However, the greater length and higher error rate of the reads generated on long-read platforms make the tools used for assembling short reads unsuitable for use in data assembly, and motivate the development of new approaches. We present LoReTTA, a tool designed for performing de novo assembly of long reads generated from viral genomes on the PacBio platform. LoReTTA exploits a reference genome to guide the assembly process, an approach that has been successful with short reads. The tool was designed to deal with reads originating from viral genomes, which feature high genetic variability, possible multiple isoforms and the dominant presence of additional organisms in clinical or environmental samples. LoReTTA was tested on a range of simulated and experimental datasets, and outperformed established long-read assemblers in terms of assembly contiguity and accuracy. The software runs under the Linux operating system, is designed for easy adaptation to alternative systems, and features an automatic installation pipeline that takes care of the required dependencies. A command-line version and a user-friendly graphical interface version are available under a GPLv3 license at https://bioinformatics.cvr.ac.uk/software/ with the manual and a test dataset

Genome sequence of human cytomegalovirus Ig-KG-H2, a variant of strain KG propagated in the presence of neutralizing antibodies

Human cytomegalovirus shed in infant urine was isolated and serially passaged in fibroblasts in the presence or absence of neutralizing antibodies. Comparison of the genome sequences of representative viruses Ig-KG-H2 (passed with antibody) and ϕ-KG-B5 (passed without antibody) revealed the presence of several mutations in each virus

Elucidating the Roles of Adaptative Polymorphisms in Modulating the Inhibitory Effects of RL13 on Human Cytomegalovirus Replication

Human cytomegalovirus (CMV), a member of the herpesvirus family, causes significant disease in immunocompromised patients and is the major infectious cause of birth defects when acquired congenitally. When CMVs in clinical samples are propagated in cell culture they rapidly acquire substitutions, frameshifts, or deletions in the RL13 gene that result in increased release of infectious virions into the culture medium. RL13 encodes RL13, a glycoprotein found in the virion envelope. How or why RL13 restricts release of cell-free virus is not known. In previous work we found that propagation of CMV in the presence of CMV-hyperimmunoglobulin (HIG) prevents mutations in RL13. However, extended passage with HIG gave rise to a virus designated Ig-KG-H2 containing additional mutations resulting four amino acid substitutions in glycoprotein M, an envelope glycoprotein involved in virion entry, two amino acid substitutions in the UL102 helicase primase subunit, and one amino acid substitution in the viral immediate early 2 (IE2) protein. As RL13 mutated slowly when Ig-KG-H2 was passaged without HIG, we hypothesized that one or more of the mutations impacting gM, UL102, or IE2 may act to modulate the inhibitory effects of RL13. In Aim 1 laboratory CMV strains derived from infectious bacterial artificial chromosome (BAC) clones were modified to encode RL13 fused to a C-terminal FLAG epitope. Small focus size and mutation of the RL13FLAG gene confirmed that RL13FLAG retains the growth impairment functions of RL13. Western blotting using a-FLAG antibody confirmed RL13FLAG expression and immunofluorescence localized RL13FLAG to large perinuclear structures that likely correspond to virion assembly compartments where virion morphogenesis occurs. In Aim 2, genetic swaps introducing sequences encoding gM from Ig- KG-H2 into two laboratory CMV strains resulted in increased focus size and preservation of RL13 during serial passage, indicating that polymorphisms in gM that arose during passage of Ig-KG-H2 with HIG function to antagonize the inhibitory effects of RL13. In Aim 3 a single H390D substitution in IE2 was shown to decrease RL13FLAG levels, increase focus size, and preserve of RL13 during serial passage, confirming that the IE2D390 polymorphism antagonizes the inhibitory effects of RL13. The same IE2D390 polymorphism was previously shown by others to permit CMV replication in the absence of UL84, a viral protein normally required for DNA replication. These results suggest two potentially independent mechanisms by which RL13 impairs CMV replication. First, interactions between RL13 and gM in vesicular membranes or within virion envelopes may alter virion morphogenesis within virion assembly compartments to promote cell-to-cell spread and reduce cell-free virus release. Second, direct or indirect interplay between RL13 and viral proteins involved in DNA replication (IE2, UL84, and perhaps UL102) may modulate the efficiency of viral DNA replication. Why CMV has evolved mechanisms to impair its replication and restrict release of cell-free virus remains uncertain; however, that in vivo CMV persists long-term in the presence of extracellular neutralizing antibodies suggests that RL13 may function to reduce unproductive release of cell-free virions since in vivo extracellular virions would be rapidly neutralized. This further suggests that in certain anatomical sites, such as kidneys or salivary glands, unknown mechanisms may actively antagonize RL13 in order to promote shedding of cell-free virions into urine or saliva and thereby transmit the infection to new hosts

The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus

Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact RL13 gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the RL13 gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13’s restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the RL13 gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in RL13 that restored replication and focus size to those of the RL13-frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of RL13 mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13’s temperance functions

The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus

Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact RL13 gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the RL13 gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13's restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the RL13 gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in RL13 that restored replication and focus size to those of the RL13-frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of RL13 mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13's temperance functions

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells

Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells