Leland F. Velicer

Biosynthesis of proteins, nucleic acids and glycosphingolipids by synchronized KB cells

Abstract The biosynthesis of glycosphingolipids and various types of proteins and nucleic acids at specific periods of the cell cycle was studied by using synchronized KB cells. Maximum incorporation of radioactive galactose, leucine and thymidine into several proteins and nucleic acids occurred as has been reported previously (6,11). Maximum incorporation of D -1[14C] galactose into glycosphingolipids was observed during the M and G-1 phases. There was a 5 fold increase in the levels of gangliosides and combined neutral glycosphingolipids during the M and G-1 phases. Thus, regulated biosynthesis of glycosphingolipids and macromolecules might be important in the cyclic expression of some of the functional properties which are characteristic of these compounds.

Transcriptional Analysis of Marek's Disease Virus Glycoprotein D, I, and E Genes: gD Expression Is Undetectable in Cell Culture

ABSTRACT The various alphaherpesviruses, including Mareks disease virus (MDV), have both common and unique features of gene content and expression. The entire MDV Us region has been sequenced in our laboratory (P. Brunovskis and L. F. Velicar, Virology 206:324–338, 1995). Genes encoding the MDV glycoprotein D (gD), glycoprotein I (gI), and glycoprotein E (gE) homologs have been found in this region, although no gG homolog was found. In this work, transcription of the tandem MDV gD, gI, and gE genes was studied and found to have both unique characteristics and also features in common with other alphaherpesviruses. MDV gD could not be immunoprecipitated from MDV GA-infected duck embryo fibroblast cells by antisera reactive to its TrpE fusion proteins, while gI and gE could be. When the gD gene was subjected to in vitro-coupled transcription-translation, the precursor polypeptide was produced and could be immunoprecipitated by anti-gD. Northern blot, reverse transcriptase PCR, and RNase protection analyses have shown that (i) no mRNA initiating directly from the gD gene could be detected; (ii) a large but low-abundance 7.5-kb transcript spanning five genes, including the one encoding gD, was seen on longer exposure; and (iii) transcription of the gI and gE genes formed an abundant bicistronic 3.5-kb mRNA, as well as an abundant 2.0-kb gE-specific mRNA. Therefore, the MDV gD gene expression is down-regulated at the transcription level in MDV-infected cell culture, which may be related to the cell-associated nature of MDV in fibroblast cells. Compared to the highly gD-dependent herpes simplex virus and the other extreme of the varicella-zoster virus which lacks the gD gene, MDV is an intermediate type of alphaherpesvirus.

Characterization of the gene encoding herpesvirus of turkeys gp57-65: comparison to Marek's disease virus gp57-65 and herpes simplex virus glycoprotein C.

A gene encoding herpesvirus of turkeys (HVT) strain FC 126 gp57-65 has been mapped to the viral genome and sequenced. The HVT (FC 126) gp57-65 gene maps toBamHI fragments K1 and M, colinear with the gene from Mareks disease virus (MDV) strain GA. HVT gp57-65 gene sequences were compared to the MDV strain GA gp57-65 gene that we sequenced previously. Overall, the two sequences are 66% identical, with greater similarity in the 3′ proximal two thirds of the genes. HVT gp57-65 gene sequences have a slightly higher overall guanosine plus cytosine (G+C) content than MDV gp57-65 gene sequences (46% vs. 41%, respectively).A single, long open reading frame capable of encoding 523 amino acids was identified within the HVT gp57-65 gene region. The predicted precursor polypeptide derived from this open reading frame would have a calculated molecular weight of 58,587. The predicted HVT gp57-65 amino acid sequences contain six potential N-linked glycosylation sites (asn-x-ser/thr). Five of these six potential N-linked glycosylation sites are conserved between the HVT and MDV predicted amino acid sequences. Hydropathic analysis of the predicted HVT gp57-65 amino acid sequences indicate the presence of an amino-terminal hydrophobic sequence, which may function as a signal peptide, and a hydrophobic carboxyl terminal sequence, which may function as a membrane anchor sequence. Overall, MDV gp57-65 and HVT gp57-65 precursor polypeptide sequences are 73% homologous and share many potential antigenic epitopes.Predicted MDV and HVT gp57-65 protein sequences are similar to those of herpes simplex virus glycoprotein C (gC) and gC-like proteins from other herpesviruses. Similarities are scattered throughout the molecule, with a primary concentration near the carboxyl half of the molecule. One stretch of 60 amino acids (HVT amino acids 378–437 and MDV amino acids 350–410) are relatively well conserved among gC-like proteins from six herpesviruses. The possible implications of these homologies and the potential roles of gC-like proteins in virus infection, growth, and replication are discussed.