Biology subjects

Azarm, K. D.

Publications and source records attributed to Azarm, K. D..

3 recordsLinked to original sources

bioRxiv2024

Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase. Author SummaryHenipaviruses, such as the prototypical Nipah virus and Hendra virus, are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, as well as for the emergent species, Ghana virus. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, application of this system to Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and identify two orally-efficacious antivirals effective against it. Further, we compare the compatibility of divergent henipavirus replicases with heterotypic viral genetic elements, providing valuable insights for how these species have evolved. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.

microbiology↗

bioRxiv2023

Structure guided mutagenesis of Henipavirus Receptor Binding Proteins reveals molecular determinants of receptor usage and antibody binding epitopes

Nipah virus (NiV) is a highly lethal, zoonotic henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, use EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, identify the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveals regions critical for GhV binding of EFNB2, and describes putative HNV antibody binding epitopes.

molecular biology↗

bioRxiv2021

Single virus assay reveals membrane determinants and mechanistic features of Sendai virus binding

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative sense RNA virus in the Paramyxoviridae family, and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic-acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to 2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that while SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nano-cluster formation. STATEMENT OF SIGNIFICANCEParamyxoviruses are a family of membrane-enveloped viruses with many notable human and animal pathogens. In this study, we develop and use an assay to observe the initial step of infection - virus binding to the host membrane - for Sendai virus, the prototypical paramyxovirus, at the single virus level. This assay uses cell membrane mimics - supported lipid bilayers - as targets for virus binding to enable easy control of the membrane components with which the virus interacts. Using our assay, we gain insight into basic biophysical questions about Sendai virus binding, including the chemical characteristics of the receptor, the cooperative nature of binding, the influence of cholesterol, and the mechanism of viral mobility following binding.

biophysics↗