Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Human herpesvirus 8 LANA interacts with proteins of the mSin3 corepressor complex and negatively regulates Epstein-Barr virus gene expression in dually infected PEL cells.

The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione S-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5xGal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.

The Epstein-Barr virus lytic transactivator Zta interacts with the helicase-primase replication proteins.

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathione S-transferase-Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses.

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.

Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.

A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.