The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the... Show moreThe sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. lmmunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays. Show less
Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor... Show moreInfection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity. Show less
Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1 ''-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The... Show moreSeveral plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1 ''-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome coronavirus (SARS-CoV) and human coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host. Show less
The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their... Show moreThe arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5' end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection. Show less
Betapapillomaviruses (beta PVs) may contribute to the aetiology of cutaneous squamous cell carcinoma. However, no high-risk types have yet been identified, possibly because the high frequency of co... Show moreBetapapillomaviruses (beta PVs) may contribute to the aetiology of cutaneous squamous cell carcinoma. However, no high-risk types have yet been identified, possibly because the high frequency of co-infection prevents a straightforward analysis of the independent effects of individual viruses. This study aimed to determine whether specific virus types were more likely to co-occur than others, thereby reducing the number of parameters needed in statistical models. Antibody data were analysed from controls who participated in case-control studies in The Netherlands, Italy and Australia and from participants in the German Nutrition Survey. Cluster analysis and two ordination techniques were used to identify patterns. Evidence of clustering was found only according to the number of viruses to which antibodies were detected. The lack of clustering of specific viral types identified suggests that if there are beta PV types that are independently related to skin carcinogenesis, they are unlikely to be identified using standard epidemiological methods. Show less
Betapapillomavirus (mu PV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an... Show moreBetapapillomavirus (mu PV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an association with cutaneous squamous cell carcinoma (SCC). Little is known about the natural history of beta PV infection and the aspects of infection that drive antibody responses. To investigate the relationship between these markers, this study assessed whether the presence or persistence of beta PV DNA in eyebrow hairs and L1 antibodies of the same beta PV type co-occurred more frequently than would be expected by chance in both a cross-sectional assessment and a longitudinal study. beta PV DNA in plucked eyebrow hairs and L1 antibodies in serum were measured in 416 participants of the Australian community-based Nambour Skin Cancer Study in 1996. Similar data were available for a subset of 148 participants in 2003. Observed co-occurrence of beta PV DNA and antibodies was compared with expected values based on prevalence. A case-wise concordance index was used to calculate the overall concordance of beta PV DNA and antibodies of the same type. No significant associations were found between the presence or persistence of beta PV DNA and antibody responses. The age and sex of the host did not influence the association, and nor did SCC status or a history of sunburns. It was concluded that beta PV antibody responses in adults are not primarily driven by beta PV infection as measured in eyebrow hairs. Other factors, such as viral load, may play a more pivotal role in the induction of detectable seroresponses. Show less
Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV... Show morePost-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV nonstructural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (01067R). Further analysis of other NS3 variants revealed phosphorylation of NS3. Show less
