No abstract

Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNAdependent RNA polymerase (3D pol ), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D pol was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D pol generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3 termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D pol initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D pol initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D pol , with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.

Seasonal variation in norovirus infection is a recognized but poorly understood phenomenon. It is likely to be based on biological, environmental and behavioural factors that regulate transmission, virulence and persistence of the virions in host populations. Understanding the seasonal dependency of norovirus infection is an important step towards understanding its epidemiology, with subsequent implementation of efficient measures of surveillance and control. Whether or not climate change could influence the seasonal patterns of norovirus infection, by impacting on its transmission, geographic distribution and prevalence, has not yet been considered. This review addresses the question.

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole} is a novel selec-Entero-and rhinoviruses are involved in a wide range of infections in humans and animals. Among the species in the enterovirus genus are the coxsackieviruses, which have been reported to be associated with various clinical manifestations, including myocarditis, pancreatitis, meningitis, and encephalitis. Other clinically relevant enteroviruses are poliovirus, which can cause paralytic poliomyelitis, and echovirus, causing aseptic meningitis or encephalomyelitis (33,36,43). Rhinoviruses are the main pathogens associated with the common cold, and, although usually mild and self-limiting, rhinovirus infections have an enormous socioeconomical impact (47,59).Enteroviruses belong to the family Picornaviridae, which consists of small, nonenveloped viruses containing a singlestranded positive-strand RNA [(ϩ)RNA] genome of 7.5 kb, which is covalently linked to a small viral protein (VPg) at its 5Ј end and polyadenylated at its 3Ј end (49). The genomic RNA has a long, highly structured 5Ј noncoding region, which contains the internal ribosome entry site, necessary for translation initiation, and a shorter 3Ј noncoding region preceding the poly(A) tract, which are both thought to be involved in RNA replication and translation (9). The coding region encodes a single polyprotein that will eventually be cleaved to generate 4 structural and 10 nonstructural proteins (either mature or in their precursor form). The icosahedral capsid of the virus is formed by 60 protomers, each one assembled by the four structural proteins, designated VP1 to -4. The nonstructural region comprises two proteases, the viral RNA-dependent RNA polymerase and seven other proteins that are involved in viral replication (8).One of the most conserved nonstructural viral proteins among picornaviruses is 2C. Although this protein is an indispensable component of the replication complex, its exact role in viral replication has remained elusive. Protein 2C appears to be multifunctional, and this entails multiple interactions (23). The amino acid sequence of 2C contains three conserved motifs which are typically found in NTP-binding proteins (motifs A and B) or in members of helicase superfamily III (motif C) (28)(29)(30)(31)69). In fact, ATPase activity has been demonstrated for several picornavirus 2C proteins (37, 57, 60) whereas so far, every attempt to demonstrate in vitro RNA helicase activity has failed. Protein 2C contains two regions involved in RNA binding (58). It has been suggested that, in poliovirus, this binding occurs at the 3Ј cloverleaf of (Ϫ)RNA (3-5); for echovirus, RNA binding was reported to occur in a nonspecific way

By screening a transcriptome database for expressed sequence tags that are specifically expressed in mammary gland and breast carcinoma, we identified a new human cytochrome P450 (CYP), termed CYP4Z1. The cDNA was cloned from the breast carcinoma line SK-BR-3 and codes for a protein of 505 amino acids. Moreover, a transcribed pseudogene CYP4Z2P that codes for a truncated CYP protein (340 amino acids) with 96% identity to CYP4Z1 was found in SK-BR-3. CYP4Z1 and CYP4Z2P genes consisting of 12 exons are localized in head-to-head orientation on chromosome 1p33. Tissue-specific expression was investigated using realtime reverse transcription PCR with normalized cDNA from 18 different human tissues. CYP4Z1 mRNA was preferentially detected in breast carcinoma tissue and mammary gland, whereas only marginal expression was found in all other tested tissues. Investigation of cDNA pairs from tumor/normal tissues obtained from 241 patients, including 50 breast carcinomas, confirmed the breast-restricted expression and showed a clear overexpression in 52% of breast cancer samples. The expression profile of CYP4Z2P was similar to that of CYP4Z1 with preference in breast carcinoma and mammary gland but a lower expression level in general. Immunoblot analyses with a specific antiserum for CYP4Z1 clearly demonstrated protein expression in mammary gland and breast carcinoma tissue specimens as well as in CYP4Z1-transduced cell lines. Confocal laser-scanning microscopy of MCF-7 cells transfected with a fluorescent fusion protein CYP4Z1-enhanced green fluorescent protein and a subcellular fractionation showed localization to the endoplasmic reticulum as an integral membrane protein concordant for microsomal CYP enzymes.