Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5′-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5′-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5′-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5′-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5′-overhang account for the observed enhanced processivity of DNA unwinding

Biochemistry and function of nidovirus replicase proteins

The order Nidovirales comprises a monophyletic group of viruses with positive-stranded RNA genomes that are classified in the families Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae. They share a conserved genome organization and a characteristic set of key replicative proteins. Although, in principle, this suggests a conserved replication mechanism, it is currently unclear how far exactly the resemblance extends on a more detailed level. This is foremost due to our poor understanding of the role of most viral proteins in the replication cycle. In addition, most of the knowledge that was obtained predominantly derives from studies of only few coronaviruses, the nidovirus subgroup with the largest known genome and therefore presumably employing the most complex replication strategy. In contrast, thus far only limited attention was given to the RNA rep_licating and processing enzymes of arteriviruses, and none at all to those of mesoni- and roniviruses, whose genome sizes are (much) smaller than those of coronaviruses. The work described in this thesis addresses some poorly or uncharacterized (domains of ) nonstructural proteins (nsps) that are likely involved in one or multiple steps of RNA replication and/or transcription of the prototypic arterivirus equine arteritis virus (EAV).UBL - phd migration 201

Interaction of viral proteins with metal ions: role in maintaining the structure and functions of viruses

Metal ions are integral part of some viral proteins and play an important role in their survival and pathogenesis. Zinc, magnesium and copper are the commonest metal ion that binds with viral proteins. Metal ions participate in maturation of genomic RNA, activation and catalytic mechanisms, reverse transcription, initial integration process and protection of newly synthesized DNA, inhibition of proton translocation (M2 protein), minus- and plus-strand transfer, enhance nucleic acid annealing, activation of transcription, integration of viral DNA into specific sites and act as a chaperone of nucleic acid. Metal ions are also required for nucleocapsid protein-transactivation response (TAR)-RNA interactions. In certain situations more than one metal ion is required e.g. RNA cleavage by RNase H. This review underscores the importance of metal ions in the survival and pathogenesis of a large group of viruses and studies on structural basis for metal binding should prove useful in the early design and development of viral inhibitors

Arterivirus replicase processing : regulatory cascade or Gordian knot?

Equine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the large replicase polyproteins pp1a and pp1ab. Processing of pp1a and pp1ab is mediated by three viral proteases of which a predicted chymotrypsin-like protease residing in nsp4 releases most non-structural proteins from the replicase polyproteins. To obtain more insight in the biochemical properties of this protease, and viral chymotrypsin-like proteases in general, the three-dimensional structure of nsp4 was determined by X-ray crystallography. The nsp4 three-dimensional structure revealed that the enzyme adopts a chymotrypsin-like fold and possesses an additional C-terminal domain (CTD) not found in most other chymotrypsin-like proteases. The structure revealed also that a connecting stretch of amino acids might facilitate movement of the CTD relative to the rest of the molecule. A site-directed mutagenesis study showed that the nsp4 CTD played a crucial role in EAV replicase processing, but that it was not required for proteloytic activity of the protease per se. Furthermore, the formation of an ion pair between Asp-119 and either Arg-4 in the N-terminus or Arg-203 in the C-terminus was suggested, which could play a role in alternate nsp4 conformations needed for e.g. different cis and trans cleavage activities. Mutations targeting the residues involved in these interactions affected the proteolytic activity of nsp4, but the data were inconclusive regarding the importance of ion pair formation. Processing of the C-terminal half of pp1a (the nsp3-8 region) by nsp4 can proceed following either of two alternative proteolytic pathways. To address the importance of both pathways, various cleavage site mutations were engineered, which were expected to block cleavage by nsp4. The experiments showed that all mutations that blocked processing of the corresponding site in the nsp3-8 precursor also blocked or severely inhibited EAV RNA synthesis. Moreover, evidence was obtained for the presence of a novel, internal nsp4 cleavage site in nsp7, which appears to be conserved among arteriviruses. The theoretical chapters in this thesis introduce the reader to (nido)viruses and nidovirus replicase maturation in particular.UBL - phd migration 201

A biochemical portrait of the nidovirus RNA polymerases and helicase

This thesis discusses the purification and activities of the SARS-coronavirus (SARS-CoV) RNA-dependent RNA polymerases (RdRps) nsp12 and nsp(7+8). The first is a large monomeric RdRp, whose stability is greatly influenced by N-terminal additions. In contrast, the latter RdRp is a remarkable hexadecameric complex that is capable of both de novo initiation and primer-extension. In addition these RNA polymerase activities, the thesis also expatiates upon the calibration of magnetic tweezers for force measurements and the dynamics of the equine arteritis virus (EAV) RNA helicase as function of NTP hydrolysis.For financial support, the Netherlands Organization for Scientific Research (NWO) is gratefully acknowledged for Toptalent grant 021.001.037 and Open Access grants 036.001.008, 036.001.250, and 036.001.619 for the publication of chapters 3, 4 and 5.UBL - phd migration 201

Antiviral resistance and persistent replication of cytomegalovirus and SARS-CoV-2 in immunocompromised patients

[eng] The main objectives of this thesis are to study the opportunistic infection caused by human cytomegalovirus and SARS-CoV-2 inquiring persistent infection and antiviral resistance. These major objectives can be further divided into: 1. Characterization of mutations associated with antiviral resistance in immunocompromised patients with refractory human cytomegalovirus infection. 1.1. Genotypic antiviral resistance testing of human cytomegalovirus target genes of conventional and new antiviral therapies by Sanger and next generation sequencing. 1.2. Phenotypic studies of uncharacterized mutations found genotypically in clinical samples by bacmid technologies, to determine the antiviral susceptibility and the replicative capacity of each individual mutation. 1.3. Searching for baseline resistant mutations before the administration of new therapies to prevent antiviral treatment failure. 1.4. Determining the incidence of cytomegalovirus antiviral resistance mutations, natural polymorphisms, and uncharacterized genetic variants in immunocompromised patients with clinically resistant cytomegalovirus infection. 2. Quantification of SARS-CoV-2 normalized viral loads in respiratory samples to study the dynamics of total viral RNA. 3. Determination of SARS-CoV-2 replicative capacity during the infection course by the presence of subgenomic RNA, and its broad applicability on the patients’ clinical monitoring. 4. Assessment of patients with persistent SARS-CoV-2 replication and/or severe COVID-19 treated with remdesivir. 5. Search of mutations associated with remdesivir failure by next-generation sequencing in severe COVID-19 patients.[spa] Esta tesis estudia la caracterización genotípica y fenotípica de mutaciones de resistencia a los anvitivirales, asi como la replicación viral persistente, en el paciente inmunocomprometido. En concreto se estudia la infección por citomegalovirus por su alta prevalencia en la población y por su alta mortalidad en pacientes imunocomprometidos, y la infección emergente por SARS-CoV-2 debido a su rápida diseminación causante de la pandemia COVID-19, la cual ha afectado especialmente a este tipo de paciente condicionando a una replicación prolongada, al uso de fármacos antivirales y la aparición de resistencias

Exploring regulatory functions and enzymatic activities in the nidovirus replicase

Members of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently known. This replicase is expressed from the viral genome by translation of two large 5__-proximal ORFs into two polyproteins, which are processed by virus-encoded proteases in 13-16 individual nonstructural proteins (nsps). The nsps direct the formation of an RNA-synthesizing complex that mediates viral genome replication, as well as the synthesis of a 3__-coterminal nested set of subgenomic (sg) mRNAs, from which the viral genes located downstream of the replicase gene are expressed. Arteriviruses and coronaviruses presumably employ a unique mechanism of discontinuous minus-strand extension to generate subgenome-length templates for sg mRNA synthesis. This thesis focused on the functional characterization of two replicase subunits and their roles in coupling different processes in the replicative cycle of equine arteritis virus (EAV), the arterivirus prototype. The biological importance of a conserved domain unique to nidoviruses (NendoU), mapping to arterivirus nsp11 and proposed to function as an endoribonuclease, was addressed. We demonstrated the recombinant nsp11 exhibits pyrimidine-specific endoribonuclease activity, and showed the critical importance of the NendoU domain for EAV RNA synthesis. In addition, we identified a multidomain replicase subunit, EAV nsp1, as a key coordinator of EAV genome replication, sg mRNA synthesis, and virus production. Our results reveal that the relative abundance of EAV mRNAs is tightly controlled by nsp1 and is critical for efficient production of new virus particles. The protein was implicated in modulating the accumulation of full-length and subgenome-length minus-strand templates for EAV mRNA synthesis. A protocol for purification of soluble recombinant nsp1, which can be used in future research on the molecular mechanisms of nsp1 function, is described.UBL - phd migration 201

Synthetic peptide studies on spike glycoprotein and 3C-like protease of the severe acute respiratory syndrome (SARS) coronavirus: perspective for SARS vaccine and drug development.

Choy Wai Yan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 98-122).Abstracts in English and Chinese.Thesis committee --- p.iStatement --- p.iiAbstract --- p.iiiAcknowledgements --- p.viGeneral abbreviations --- p.viiiAbbreviations of chemicals --- p.xTable of contents --- p.xiList of figures --- p.xvList of tables --- p.xviiiChapter 1 --- Introduction --- p.1Chapter 1.1 --- Severe acute respiratory syndrome (SARS) - An overview --- p.1Chapter 1.1.1 --- Epidemiology of SARS --- p.1Chapter 1.1.2 --- Clinical presentation of SARS --- p.2Chapter 1.1.3 --- Diagnostic tests of SARS --- p.5Chapter 1.1.4 --- Treatment of SARS --- p.7Chapter 1.2 --- Severe acute respiratory syndrome coronavirus (SARS- CoV) --- p.8Chapter 1.2.1 --- The etiological agent of SARS --- p.8Chapter 1.2.2 --- The coronaviruses --- p.9Chapter 1.2.3 --- Genome of SARS-CoV --- p.11Chapter 1.3 --- Spike (S) glycoprotein of SARS-CoV --- p.14Chapter 1.3.1 --- Functions of SARS-CoV S glycoprotein --- p.15Chapter 1.3.2 --- Receptors for S glycoprotein of SARS-CoV --- p.17Chapter 1.4 --- 3C-like protease (3CLPro) of SARS-CoV --- p.20Chapter 1.4.1 --- Extensive proteolytic processing of SARS-CoV replicase polyproteins --- p.20Chapter 1.4.2 --- SARS-CoV 3CLPro --- p.21Chapter 1.4.3 --- Substrate specificity of SARS-CoV 3CLPro --- p.22Chapter 1.5 --- Combating SARS - Vaccine and drug development --- p.24Chapter 1.5.1 --- Vaccine development against SARS --- p.24Chapter 1.5.2 --- Drug development against SARS --- p.25Chapter 1.6 --- Project objectives of this thesis --- p.27Chapter 1.6.1 --- Synthetic Peptide Studies on SARS-CoV S glycoprotein --- p.27Chapter 1.6.2 --- Synthetic Peptide Studies on SARS-CoV 3CLPro --- p.28Chapter 2 --- Materials and Methods --- p.30Chapter 2.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.30Chapter 2.1.1 --- Bioinformatics analyses of SARS-CoV S gly- coprotein --- p.30Chapter 2.1.2 --- Peptide design and molecular modeling --- p.32Chapter 2.1.3 --- Solid phase peptide synthesis (SPPS) --- p.33Chapter 2.1.4 --- Peptide conjugation --- p.35Chapter 2.1.5 --- Immunization in rabbits and monkeys --- p.36Chapter 2.1.6 --- ELISA analysis --- p.37Chapter 2.1.7 --- Immunofluorescent confocal microscopy --- p.39Chapter 2.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.40Chapter 2.2.1 --- Protein expression and purification --- p.40Chapter 2.2.2 --- Solid phase peptide synthesis (SPPS) --- p.41Chapter 2.2.3 --- Peptide cleavage assay --- p.44Chapter 2.2.4 --- Molecular docking --- p.46Chapter 3 --- Results --- p.48Chapter 3.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.48Chapter 3.1.1 --- General features and structural analyses of the S glycoprotein --- p.48Chapter 3.1.2 --- Peptides design and synthesis --- p.53Chapter 3.1.3 --- ELISA analysis and immunofluorescent con- focal microscopy --- p.55Chapter 3.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.62Chapter 3.2.1 --- Substrate specificity of SARS-CoV 3CLPro . . --- p.62Chapter 3.2.2 --- Molecular docking of SARS-CoV 3CLPro and peptide substrates --- p.74Chapter 4 --- Discussion --- p.78Chapter 4.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.78Chapter 4.1.1 --- Synthetic peptides elicited SARS-CoV specific antibodies --- p.78Chapter 4.1.2 --- Factors affecting the specificity and antigenic- ity of synthetic peptides --- p.80Chapter 4.1.3 --- Next step towards vaccine development --- p.83Chapter 4.1.4 --- A synthetic peptide-based approach --- p.84Chapter 4.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.86Chapter 4.2.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.87Chapter 4.2.2 --- Sequence comparison between SARS-CoV 3CLpro cleavage sites --- p.90Chapter 4.2.3 --- A rapid and high throughput approach to screen protease substrate specificity --- p.94Bibliography --- p.9

Biotechnology to Combat COVID-19

This book provides an inclusive and comprehensive discussion of the transmission, science, biology, genome sequencing, diagnostics, and therapeutics of COVID-19. It also discusses public and government health measures and the roles of media as well as the impact of society on the ongoing efforts to combat the global pandemic. It addresses almost every topic that has been studied so far in the research on SARS-CoV-2 to gain insights into the fundamentals of the disease and mitigation strategies. This volume is a useful resource for virologists, epidemiologists, biologists, medical professionals, public health and government professionals, and all global citizens who have endured and battled against the pandemic