Human parechoviruses (HPeV), members of the Parechovirus genus of Picornaviridae, are frequent pathogens but have been comparatively poorly studied, and little is known of their diversity, evolution, and molecular biology. To increase the amount of information available, we have analyzed 7 HPeV strains isolated in California between 1973 and 1992. We found that, on the basis of VP1 sequences, these fall into two genetic groups, one of which has not been previously observed, bringing the number of known groups to five. While these correlate partly with the three known serotypes, two members of the HPeV2 serotype belong to different genetic groups. In view of the growing importance of molecular techniques in diagnosis, we suggest that genotype is an important criterion for identifying viruses, and we propose that the genetic groups we have defined should be termed human parechovirus types 1 to 5. Complete nucleotide sequence analysis of two of the Californian isolates, representing two types, confirmed the identification of a new genetic group and suggested a role for recombination in parechovirus evolution. It also allowed the identification of a putative HPeV1 cis-acting replication element, which is located in the VP0 coding region, as well as the refinement of previously predicted 5 and 3 untranslated region structures. Thus, the results have significantly improved our understanding of these common pathogens.

SummaryThe controlled release of RNA polymerase II (RNAPII) from promoter-proximal pausing (PPP) sites is critical for transcription elongation in metazoans. We show that the human tumor suppressor BRCA2 interacts with RNAPII to regulate PPP release, thereby preventing unscheduled RNA-DNA hybrids (R-loops) implicated in genomic instability and carcinogenesis. BRCA2 inactivation by depletion or cancer-causing mutations instigates RNAPII accumulation and R-loop accrual at PPP sites in actively transcribed genes, accompanied by γH2AX formation marking DNA breakage, which is reduced by ERCC4 endonuclease depletion. BRCA2 inactivation decreases RNAPII-associated factor 1 (PAF1) recruitment (which normally promotes RNAPII release) and diminishes H2B Lys120 ubiquitination, impeding nascent RNA synthesis. PAF1 depletion phenocopies, while its overexpression ameliorates, R-loop accumulation after BRCA2 inactivation. Thus, an unrecognized role for BRCA2 in the transition from promoter-proximal pausing to productive elongation via augmented PAF1 recruitment to RNAPII is subverted by disease-causing mutations, provoking R-loop-mediated DNA breakage in BRCA2-deficient cells.

Coxsackievirus A9 (CAV9), a member of the Enterovirus genus of Picornaviridae, is a common human pathogen and is one of a significant number of viruses containing a functional arginine-glycine-aspartic acid (RGD) motif in one of their capsid proteins. Previous studies identified the RGD-recognizing integrin ␣ v ␤ 3 as its cellular receptor. However, integrin ␣ v ␤ 6 has been shown to be an efficient receptor for another RGDcontaining picornavirus, foot-and-mouth disease virus (FMDV). In view of the similarity in sequence context of the RGD motifs in CAV9 and FMDV, we investigated whether ␣ v ␤ 6 can also serve as a receptor for CAV9. We found that CAV9 can bind to purified ␣ v ␤ 6 and also to SW480 cells transfected with ␤ 6 cDNA, allowing expression of ␣ v ␤ 6 on their surface, but it cannot bind to mock-transfected cells. In addition, a higher yield of CAV9 was obtained in ␤ 6 -expressing cells than in mock-transfected cells. There was no similar enhancement in infection with an RGD-less CAV9 mutant. We also found ␤ 6 on the surface of GMK cells, a cell line which CAV9 infects efficiently by an RGD-dependent mechanism. Significantly, this infection is blocked by an antibody to ␣ v ␤ 6 , while this antibody did not block the low level of infection by the RGD-less mutant. Thus, integrin ␣ v ␤ 6 is an RGD-dependent receptor for CAV9 and may be important in natural CAV9 infections.Coxsackievirus A9 (CAV9), a member of the Enterovirus genus of the Picornaviridae family, is a common human pathogen that causes central nervous system infections and myocarditis as well as a range of milder illnesses (11, 43). The picornavirus particle is about 28 nm in diameter and consists of a naked capsid with icosahedral symmetry, surrounding a positive sense RNA genome of around 7,100 to 8,500 nucleotides (41). The capsid is made up of 60 copies of each of four proteins, VP1 to VP4, and interacts with receptors during the early stages of infection. A number of picornavirus receptors have been identified, and they include several integrins (9). Integrins are named for their role in integrating the intracellular cytoskeleton with the extracellular matrix and participate in a number of cell-cell, cell-matrix interactions. They are heterodimeric proteins with one ␣ and one ␤ subunit, and each contains an exodomain, the site of interaction with ligands, a transmembrane region, and a cytoplasmic domain. The 8 known ␤ subunits and 14 known ␣ subunits combine to give at least 21 different heterodimeric combinations (16,38,40).The arginine-glycine-aspartic acid (RGD) motif was the first integrin-binding sequence to be identified (38) and is recognized by several integrins (␣

Human parechoviruses (HPeVs), members of the family Picornaviridae, are classified into six types. To investigate the dynamics and likelihood of recombination among HPeVs, we compared phylogenies of two distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3-5) and prototype sequences (types 1-6). Evidence for frequent recombination between HPeV1, 4, 5 and 6 was found. The likelihood of recombination was correlated with the degree of VP1 divergence and differences in isolation dates, both indicative of evolutionary times of divergence. These temporal dynamics were found to be most similar to those of human enterovirus species B variants. In contrast, HPeV3 remained phylogenetically distinct from other types throughout the genome. As HPeV3 is equally divergent in nucleotide sequence from the other HPeV types, its genetic isolation may reflect different biology and changed cellular tropisms, arising from the deletion of the RGD motif, and likely use of a non-integrin receptor.