Robert J. Geraghty

Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B

Virus-induced membrane fusion can be subdivided into three phases defined by studies of class I and class II fusion proteins. During Phase I, two membranes are brought into close apposition. Phase II marks the mixing of the outer membrane leaflets leading to formation of a hemifusion intermediate. A fusion pore stably forms and expands in Phase III, thereby completing the fusion process. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins to complete membrane fusion, but none has been defined as class I or II. Therefore, we investigated whether HSV-1-induced membrane fusion occurred following the same general phases as those described for class I and II proteins. In this study we demonstrate that glycoprotein D (gD) and the glycoprotein H and glycoprotein L complex (gHL) mediated lipid mixing indicative of hemifusion. However, content mixing and full fusion required glycoprotein B (gB) to be present along with gD and gHL. Our results indicate that, like class I and II fusion proteins, fusion mediated by HSV-1 glycoproteins occurred through a hemifusion intermediate. In addition, both gB and gHL are probably directly involved in the fusion process. From this, we propose a sequential model for fusion via HSV-1 glycoproteins whereby gD is required for Phase I, gHL is required for Phase II, and gB is required for Phase III. We further propose that glycoprotein H and gB are likely to function sequentially to promote membrane fusion in other herpesviruses such as Epstein–Barr virus and human herpesvirus 8.

Cell type-dependence for Vpu function

Human immunodeficiency virus type 1 Vpu has been shown to facilitate virus release from HeLa cells. We demonstrated that Vpu expression is not required for efficient virus release from Cos 1 and CV‐1 cells. A yeast GAL4 transcriptional activation system was used to screen for cellular proteins that may interact with Vpu. One such protein was identified which we provisionally designate “Vpu interactive protein” or VIP.

The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus.

C7-Substituted 2-hydroxyisoquinoline-1,3-diones inhibit the strand transfer of HIV integrase (IN) and the reverse-transcriptase-associated ribonuclease H (RNH). Hepatitis C virus (HCV) NS5B polymerase shares a similar active site fold to RNH and IN, suggesting that N-hydroxyimides could be useful inhibitor scaffolds of HCV via targeting the NS5B. Herein we describe the design, chemical synthesis, replicon and biochemical assays, and molecular docking of C-6 or C-7 aryl substituted 2-hydroxyisoquinoline-1,3-diones as novel HCV inhibitors. The synthesis involved an improved and clean cyclization method, which allowed the convenient preparation of various analogs. Biological studies revealed that the C-6 analogs, a previously unknown chemotype, consistently inhibit both HCV replicon and recombinant NS5B at low micromolar range. Molecular modeling studies suggest that these inhibitors may bind to the NS5B active site.

Contributions of gD receptors and glycosaminoglycan sulfation to cell fusion mediated by herpes simplex virus 1

Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

Background: The severe acute respiratory syndrome (SARS) virus has undergone mutations in its receptor-binding domain. Results: We used biochemical, functional, and crystallographic methods to investigate these mutations. Conclusion: These mutations were viral adaptations to either the human or palm civet receptor. Significance: This research elucidates detailed mechanisms of host receptor adaptation by the SARS virus and can help predict and monitor future evolution of the SARS virus in animals. The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

A Virus-Binding Hot Spot on Human Angiotensin-Converting Enzyme 2 Is Critical for Binding of Two Different Coronaviruses

ABSTRACT How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

Hydroxamic acids block replication of hepatitis C virus.

Intrigued by the role of protein acetylation in hepatitis C virus (HCV) replication, we tested known histone deacetylase (HDAC) inhibitors and a focused library of structurally simple hydroxamic acids for inhibition of a HCV subgenomic replicon. While known HDAC inhibitors with varied inhibitory profiles proved to be either relatively toxic or ineffective, structure-activity relationship (SAR) studies on cinnamic hydroxamic acid and benzo[b]thiophen-2-hydroxamic acid gave rise to compounds 22 and 53, which showed potent and selective anti-HCV activity and therefore are promising starting points for further structural optimization and mechanistic studies.

Mutagenic analysis of herpes simplex virus type 1 glycoprotein L reveals the importance of an arginine-rich region for function

Herpes simplex virus type 1 (HSV-1) glycoproteins H and L (gH and gL) are required for virus-induced membrane fusion. Expression of gH at the virion or infected cell surface is mediated by the chaperone-like activity of gL. We have previously shown that a region between amino acids 155 and 161 is critical for gL chaperone-like activity. Here, we conducted Ala substitution mutagenesis of residues in this region and found that substitution of Cys160, Arg156, Arg158, or Arg156/158/159 with Ala resulted in a gL mutant that bound gH but displayed a reduced ability in gH trafficking and membrane fusion. Substitution of Arg156 with another positively charged amino acid, Lys, restored function. Substitution of Arg158 with Lys restored function in gH trafficking and cell fusion but not virus entry. These results indicate that an arginine-rich region of gL is critical for function.

Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage

ABSTRACT The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.

Synthesis and antiviral evaluation of 4′-(1,2,3-triazol-1-yl)thymidines

Non-obligate chain terminating nucleosides with a linear substituent (azido or ethynyl group) at the 4′ position represent an important class of compounds in antiviral discovery, particularly against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). We have previously shown that 3′-azidothymidine (AZT)-derived 1,2,3-triazoles can be potent inhibitors of HIV-1. To gauge the medicinal chemistry impact of functionalizing the 4′-linear substituent and possibly generate novel antiviral nucleoside scaffolds, we have explored azide–alkyne cycloaddition reactions with 4′-azidothymidine (ADRT). The Ru-mediated reaction failed and the Cu-catalyzed variant generated 1,2,3-triazoles (9a–y) with only modest yields and efficiencies, indicating a substantial steric barrier around the 4′-azido group. Antiviral screening identified a few triazole analogues moderately active against HIV-1 (18–62% inhibition at 10 μM) and/or influenza A virus (15–50% inhibition at 10 μM), and none active against West Nile virus (WNV) or HCV. These results suggest that the linear 4′ azido group of ADRT may be essential for target binding and that its chemical manipulation could largely compromise antiviral potency.