Homologous and heterologous interference requires bovine herpesvirus-1 glycoprotein D at the cell surface during virus entry.

Expression of glycoprotein D (gD) of alphaherpesviruses protects cells from superinfection by homologous and heterologous viruses by a mechanism termed interference. We recently showed that MDBK cells expressing bovine herpesvirus (BHV)-1gD (MDBK(gD)) resist BHV-1, pseudorabies virus (PRV) and herpes simplex virus-1 (HSV-1) but not the more closely related BHV-5 infection as determined by the number of plaques produced. However, the plaque size is reduced in all four viral infections suggesting a block in cell-to-cell transmission. Here, we show that MDBK cells expressing truncated BHV-1 gD, designated MDBK(t-gD), secreted soluble gD and were fully susceptible to infection by all the four viruses when the cells were washed prior to infection. When MDBK cells or MDBK(t-gD) cells were treated with medium containing truncated gD prior to infection, they partially resisted BHV-1, PRV and HSV-1 but not BHV-5. Interestingly, both BHV-1 and BHV-5 formed normal-sized plaques in MDBK(t-gD) cells suggesting that the viruses were able to spread efficiently. Thus BHV-1 gD is required at the cell surface at the time of infection in order to block BHV-1, HSV-1 and PRV infections, consistent with a common coreceptor for the three gDs.

Bovine herpesvirus type 2 is closely related to the primate alphaherpesviruses.

Bovine herpesvirus type 2 (BoHV-2), also known as bovine mammillitis virus, is classified in the Family Herpesviridae, Subfamily Alphaherpesvirinae, and Genus Simplexvirus along with herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) and other primate simplexviruses on the basis of similarities in 4 genes within the 15 kb U(L) 23-29 cluster. This could be explained either by a global similarity or a recombination event that brought primate herpesviral sequences into a bovine virus. Our sequences for DNA polymerase (U(L)30), a large gene adjacent to the previously identified conserved cluster, and glycoprotein G (U(S)4), a gene as distant from the cluster as possible on the circularized genome, confirm the close relationship between BoHV-2 and the primate simplexviruses, and argue for a global similarity and probably a close evolutionary relationship. Thus one can speculate that BoHV-2 may represent a greater hazard to humans than has been appreciated previously.

Cloning and functional studies of a novel gene aberrantly expressed in RB-deficient embryos.

The tumor suppressor RB regulates diverse cellular processes such as G1/S transition, cell differentiation, and cell survival. Indeed, Rb-knockout mice exhibit phenotypes including ectopic mitosis, defective differentiation, and extensive apoptosis in the neurons. Using differential display, a novel gene, Rig-1, was isolated based on its elevated expression in the hindbrain and spinal cord of Rb-knockout embryos. The longest open reading frame of Rig-1 encoded a polypeptide that consists of a putative extracellular segment with five immunoglobulin-like domains and three fibronectin III-like domains, a putative transmembrane domain, and a distinct intracellular segment. The Rig-1 sequence was 40% identical to the recently identified roundabout protein. Consistent with the predicted transmembrane nature of the protein, Rig-1 protein was present in the membranous fraction. Antisera raised against the putative extracellular and intracellular segments of Rig-1 reacted with an approximately 210-kDa protein in mouse embryonic CNS. Rig-1 mRNA was transiently expressed in the embryonic hindbrain and spinal cord. Elevated levels of Rig-1 mRNA and protein were found in Rb-/- embryos. Ectopic expression of a transmembrane form of Rig-1, but not the secreted form, promoted neuronal cell entrance to S phase and repressed the expression of a marker of differentiated neuron, Talpha1 tubulin. Thus Rig-1, a possible distant relative of roundabout, may mediate some of the pleiotropic roles of RB in the developing neurons.

Cellular expression of bovine herpesvirus 1 gD inhibits cell-to-cell spread of two closely related viruses without blocking their primary infection.

Alphaherpesviral glycoprotein D (gD) is a critical component of the cell membrane penetration system. Cells that express gD of herpes simplex virus type 1 (HSV1), pseudorabies virus (PRV), or bovine herpesvirus type 1.1 (BHV1.1) resist infection by the homologous virus due to interference with viral entry at the level of penetration. BHV1.1 gD interferes with the distantly related viruses HSV1 and PRV despite only a 30-40% sequence similarity and the complete absence of antigenic cross-reactivity among the three gDs. The six cysteines that form three intrachain disulfide bonds in HSV1 are also present in PRV and BHV1.1 gD, suggesting structural similarities among the gD homologs. Functional similarities were postulated to be responsible for cross-interference. To test this hypothesis, we constructed a BHV1.1 gD-expressing cell line (MDBKgD) and assessed its resistance to the homologous BHV1.1 and two closely related viruses, BHV1.2 and BHV5. The gDs of these viruses share 98. 3% and 86% amino acid identity with BHV1.1 gD and bound monoclonal antibodies directed against all five neutralizing epitopes mapped on BHV1.1 gD. MDBKgD cells were resistant to BHV1.1 but fully susceptible to BHV1.2 and BHV5 infection as measured by plaque numbers and single cycle growth kinetics. However, all three viruses, but not vesicular stomatitis virus, made smaller plaques on MDBKgD cells than on control cells. These data suggest that gD-mediated interference is expressed both at the level of initial infection and at the level of cell-to-cell spread and that these two levels can be distinguished by using closely related viruses.

DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis.

Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation. Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast. In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control. DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals. Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells. This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells. In addition, mammalian DSBR proteins and their activities are discussed. An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated. Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis.