Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.

Structures of the compact helical core domains of feline calicivirus and murine norovirus VPg proteins.

We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

Development of an optimized RNA-based murine norovirus reverse genetics system.

Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of in vitro transcribed capped RNA directly into cells. The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.

Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence.

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.

Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea.

BACKGROUND: Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of L. hongkongensis. In this study, a multilocus sequence typing (MLST) system was developed for L. hongkongensis. The system was used to characterize 146 L. hongkongensis isolates, including 39 from humans and 107 from fish. RESULTS: Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (ilvC) to 45 (thiC)]. All seven genes showed very low d(n)/d(s) ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (I(S)(A)) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The I(S)(A) for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (acnB) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the rho, acnB and thiC loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of L. hongkongensis. A website for L. hongkongensis MLST was set up and can be accessed at http://mlstdb.hku.hk:14206/MLST_index.html. CONCLUSION: A highly reproducible and discriminative MLST system was developed for L. hongkongensis.

MP1 homologue-based multilocus sequence system for typing the pathogenic fungus Penicillium marneffei: a novel approach using lineage-specific genes.

A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of Penicillium marneffei, the most important thermal dimorphic fungus causing respiratory, skin, and systemic mycosis in Southeast Asia. The sequences of 11 housekeeping genes were identical among 10 strains of P. marneffei, but those of MP1 and its 13 homologues, a novel superfamily of mannoproteins in the subdivision Pezizomycotina of Ascomycetes, mostly species of Penicillium and Aspergillus, showed significant variations. Therefore, a multilocus sequence typing (MLST) system for P. marneffei was constructed using MP1 (549 bp) and the four of its homologues (MPLP4 [337 bp], MPLP7 [347 bp], MPLP10 [546 bp], and MPLP13 [422 bp]) that showed the greatest variations. Among the 2,201 bp of the five loci, 183 polymorphic sites were observed in 44 strains of P. marneffei. The median number of alleles at each locus was five (range, 5 [MPLP4, MPLP7, and MPLP13] to 15 [MPLP10]). Four of the five genes had nonsynonymous substitution/synonymous substitution (d(n)/d(s)) ratios of >1. A total of 35 different sequence types (STs) were assigned to the 44 P. marneffei isolates, with 28 of the 35 STs identified only once. The discriminatory power was 0.9884. MP1 and its homologues were better than housekeeping genes for MLST in P. marneffei. Due to their more rapid evolutionary rates, lineage-specific genes may be better candidates than housekeeping genes for sequence-based typing, especially in microbes that evolve slowly or have evolved recently.

In silico analysis of 16S ribosomal RNA gene sequencing-based methods for identification of medically important anaerobic bacteria.

This study is the first study that provides useful guidelines to clinical microbiologists and technicians on the usefulness of full 16S rRNA sequencing, 5'-end 527-bp 16S rRNA sequencing and the existing MicroSeq full and 500 16S rDNA bacterial identification system (MicroSeq, Perkin-Elmer Applied Biosystems Division, Foster City, California, USA) databases for the identification of all existing medically important anaerobic bacteria. Full and 527-bp 16S rRNA sequencing are able to identify 52-63% of 130 Gram-positive anaerobic rods, 72-73% of 86 Gram-negative anaerobic rods and 78% of 23 anaerobic cocci. The existing MicroSeq databases are able to identify only 19-25% of 130 Gram-positive anaerobic rods, 38% of 86 Gram-negative anaerobic rods and 39% of 23 anaerobic cocci. These represent only 45-46% of those that should be confidently identified by full and 527-bp 16S rRNA sequencing. To improve the usefulness of MicroSeq, bacterial species that should be confidently identified by full and/or 527-bp 16S rRNA sequencing but not included in the existing MicroSeq databases should be included.

Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada.

Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37 degrees C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences from Clostridium propionicum, Clostridium neopropionicum, and Atopobium minutum, respectively. The G+C content of strain HKU16T is 40.2% +/- 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species, Catabacter hongkongensis gen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family, Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility of C. hongkongensis. Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.