Margaret M. Willcocks

Application of electronmicroscopy, enzyme immunoassay, and RT‐PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit

During 1997, an extensive outbreak of astrovirus occurred in a unit where paediatric patients were being treated for leukaemias and inherited immune deficiency disorders. Prolonged shedding of virus for many months following infection was demonstrated in three patients who had undergone bone marrow transplantation. Comparison of reverse transcription‐polymerase chain reaction (RT‐PCR), enzyme immunoassay (EIA), and electronmicroscopy (EM) to monitor the outbreak showed that many subclinical infections, mainly in children aged > 3 years could only be detected by RT‐PCR. Use of RT‐PCR revealed that several patients were infected earlier and shed virus for longer than by using EM or EIA. The virus responsible for the outbreak was identified as HAstV‐1 and was shown to have a sequence that differed from a strain obtained in 1988. J. Med. Virol. 57:313–321, 1999.

Defective translation of measles virus matrix protein in a subacute sclerosing panencephalitis cell line

Subacute sclerosing panencephalitis (SSPE) is a slowly progressing fatal human disease of the central nervous system (CNS) that is associated with measles virus persistence. Virus nucleocapsids are present in the brain1,2 and the patient is in a state of hyperimmunization towards this agent. However, although all other structural polypeptides are recognized by the immune system, there is a markedly decreased antibody response towards virus matrix or membrane protein3,4. Matrix protein has not been detected in brain cells5 and infectious virus is not present. The absence of this virus structural polypeptide is thought to account for the apparent restriction in virus maturation both in vivo and in vitro. SSPE viruses can only rarely be rescued from brain tissue by co-cultivation or cell fusion techniques using tissue culture cell lines susceptible to measles virus infection6. Often this procedure fails to yield a lytic budding virus but produces instead a carrier cell line in which the agent is cell associated. These lines (known as SSPE cell lines) also do not contain matrix protein7,8. However, the reason for this deficiency is unknown. We have therefore now examined an SSPE cell line which does not yield infectious virus in order to define this process further. We found that although messenger RNA for membrane protein was present, it was unable to form normal matrix protein in translation reactions.

Molecular Epidemiology of Childhood Astrovirus Infection in Child Care Centers

This study assessed the role of human astrovirus (HAstV) in outbreaks and sporadic cases of diarrhea among children attending child care centers (CCCs) and determined the infecting astrovirus antigenic types by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis. Eight astrovirus outbreaks occurred in 6 CCCs. Of 179 children with diarrhea, 36 (20%) had astrovirus-associated diarrhea. Diarrhea stools obtained during diarrhea outbreaks were more likely to contain astrovirus (40/476) than were samples not associated with a diarrhea outbreak (14/452) (P<.001). Type-specific RT-PCR and DNA sequencing identified 5 outbreaks associated with HAstV-1 and 3 outbreaks with HAstV-2. Sequential outbreaks in 2 CCCs occurred with a different type in the same year. Phylogenetic analysis identified 6 clades of HAstV-1 and 2 clades of HAstV-2 during this 1-year surveillance. Astrovirus was a significant cause of diarrhea outbreaks, and 2 antigenic types were present in the community during 1 diarrhea season.

Relationships between Monoclonal Antibody-binding Sites on the Measles Virus Haemagglutinin

Twenty-one monoclonal antibodies directed against the measles virus haemagglutinin have recently been obtained. These were known to fall into five groups, each defined by its effects on the biological functions of the H protein. A representative of each group was selected and examined by competitive radioimmunoassay in an attempt to deduce the relationships between antibody-binding sites on the antigen. It was found that these five antibodies formed three binding groups which recognized different but overlapping areas of the molecule. These three areas formed a series of sites which traversed the active region of the H polypeptide. A haemagglutinin-directed monoclonal antibody which displayed a haemolysin-inhibiting activity was also examined. This antibody fitted into the binding group scheme determined here and there was no additional binding site for this molecule on the F protein itself.

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

ABSTRACT Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5′ untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3′ end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES

ABSTRACT The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId2 domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.

Processing and intracellular location of human astrovirus non-structural proteins.

Human astrovirus (HAst) non-structural polyproteins are encoded in two open reading frames linked in expression by a ribosomal frameshifting event. The first of these (ORF 1a) specifies the serine protease, whilst the second (ORF 1b) encodes the virus RNA-dependent RNA polymerase. The ORF 1a product contains an unusual motif for small RNA viruses which could potentially direct proteins to the cell nucleus. We have expressed part of ORF 1a containing this motif and the whole of ORF 1b separately in recombinant baculovirus and raised specific antisera to each. We now report that expressed proteins from ORF 1a accumulate in the nucleus of both baculovirus-infected insect cells and HAst-infected CaCo-2 cells. In contrast the products of ORF 1b remain predominantly cytoplasmic.

Comparison of Lytic and Persistent Measles Virus Matrix Proteins by Competition Radioimmunoassay

Measles virus matrix (M) proteins were compared by competitive monoclonal antibody-binding studies. Three strains of measles and of subacute sclerosing panencephalitis viruses were found to be identical in this way. The matrix protein formed by Edmonston strain virus during a persistent infection could be distinguished from that made in the lytic virus infection. It is concluded that structural alterations in the M peptide can occur during persistence.

Functional analysis of Kaposi's sarcoma–associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation

Kaposis sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.

Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage

ABSTRACT In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6Pro. This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.