An ORF-2a protein is not present at a significant level in virions of the arterivirus lactate dehydrogenase-elevating virus.

MRNA2 of the arteriviruses lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV) encodes two proteins that are read in different frames, an about 26 kDa minor envelope glycoprotein and an about 8 kDa protein that lacks N-glycosylation sites and a signal peptide, but possesses a central hydrophobic segment. Recent studies have shown that both proteins of EAV are translated from mRNA 2 in EAV infected BHK cells, that the 8 kDa protein is membrane associated and that small amounts of it are recovered in purified virions (Snijder, E.J., van Tol, H., Pederson, K.W., Raamsman, M.J.B., de Vries, A.A.F., 1999. Identification of a novel structural protein of arteriviruses. J. Virol. 73, 6335-6345). The authors concluded that the 8 kDa protein is another arterivirus envelope protein and designated it E protein. However, we have not detected a significant level of an 8 kDa protein in LDV virions and thus conclude that it is not a structural virion component.

Isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization.

Lactate dehydrogenase-elevating virus (LDV) was first identified as a contaminant of transplantable mouse tumors that were passaged in laboratory mice. It has been assumed that these LDVs originated from LDVs endemic in wild house mouse populations. In order to test this hypothesis and to explore the relationships between LDVs from wild house mice among each other and to those isolated from laboratory mice, we have isolated LDVs from wild house mice and determined their biological and molecular properties. We have screened for LDV tissues of 243 wild house mice that had been caught in various regions of North, Central and South America between 1985 and 1994. We were able to isolate LDVs from the tissues of four mice, three had been caught in Baltimore, MD and one in Montana. We demonstrate that the phenotypic properties (ability to establish a long-term viremic infection, low immunogenicity of the neutralization epitope, high resistance to antibody neutralization and lack of neuropathogenicity) of the four wild house mouse LDVs are identical to those of the primary LDVs isolated from transplantable tumors (LDV-P and LDV-vx), which are distinct from those of the neuropathogenic LDV-C. Furthermore, ORF 5 and ORF 2 and their protein products (the primary envelope glycoprotein VP-3P, and the minor envelope glycoprotein, respectively) of the wild house mouse LDVs were found to be closely related to those of LDV-P and LDV-vx. The LDVs caught in Baltimore, MD were especially closely related to each other, whereas the LDV isolated in Montana was more distantly related, indicating that it had evolved independently. The ectodomain of VP-3P of all four wild house mouse LDVs, like those of LDV-P and LDV-vx, possess the same three polylactosaminoglycan chains, two of which are lacking in the VP-3P ectodomain of LDV-C. These results further strengthen the conclusion that the three polylactosaminoglycan chains are the primary determinants of the phenotypic properties of LDV-P/vx.

Detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and RT-PCR assays and its removal from the tumor cell.

It is known that lactate dehydrogenase-elevating virus (LDV) of mice is a common contaminant of transplantable tumors of both murine and human origin. It is imperative that tumors that are maintained by transplantation in mice are examined for LDV and freed of the virus, when present, before use in experimental studies, because an LDV infection of mice exerts considerable effects on lymphoid cell populations and cytokine production and other effects. Methods for LDV detection are described using a biological assay and reverse transcription (RT)-polymerase chain reaction (PCR) technology and their application is illustrated. A differential RT-PCR method that distinguishes between three quasispecies of LDV is also described and applied to an examination of LDVs isolated from a number of different tumors. Each of the LDV isolates was found to contain at least two different quasispecies, generally in different concentrations.

Membrane association of the C-terminal half of the open reading frame 1a protein of lactate dehydrogenase-elevating virus.

ORF 1a of lactate dehydrogenase-elevating virus, strain P (LDV-P), encodes a protein of 2206 amino acids. Eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the C-terminal half of the molecule (amino acids 980-1852) that flank the serine protease domain. cDNAs encoding ORF 1a protein segments encompassing transmembrane segments 5 to 11 and its amphipathic C-terminal end as well as the N-terminal 80 amino acids of the downstream ORF 1b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. The synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. When synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. Furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, pH 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase K. No N-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. The results indicate that the C-terminal half of the ORF 1a protein represents a non-glycosylated integral membrane protein. Potential modes of synthesis and function of the protein are discussed. In addition, the results showed that the synthesis of the ORF 1a protein was generally terminated at its termination codon, but that read-through into the ORF 1b gene occurred with low frequency.

Differential glycosylation of the ectodomain of the primary envelope glycoprotein of two strains of lactate dehydrogenase-elevating virus that differ in neuropathogenicity.

ORF 5 encoding the primary envelope glycoprotein, VP-3P, of a highly neuropathogenic isolate of lactate dehydrogenase-elevating virus (LDV-v) has been sequenced. It exhibits 92% nucleotide identity with the ORF 5 of an LDV isolate that lacks neuropathogenicity, LDV-P, and the amino acid identities of the predicted VP-3Ps of the two strains is 90%. Most striking, however, is the absence in the ectodomain of LDV-v VP-3P of two out of three potential N-glycosylation sites present in the ectodomain of VP-3P of LDV-P. The ectodomain of VP-3P has been implicated to play an important role in host receptor interaction. VP-3P of another neuropathogenic LDV strain, LDV-C, lacks the same two N-glycosylation sites (Godeny et al., 1993). In vitro transcription/translation of the ORFs 5 of LDV-P and LDV-v indicated that all three N-glycosylation sites in the ectodomain of LDV-P VP-3P became glycosylated when synthesized in the presence of microsomal membranes, whereas the glycosylation of the ORF 5 proteins of LDV-v and LDV-C was consistent with glycosylation at a single site. No other biological differences between the neuropathogenic and non-neuropathogenic strains have been detected. They replicate with equal efficiency in mice and in primary macrophage cultures.

The envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography.

We have studied the membrane topography and N-glycosylation of the envelope proteins of lactate dehydrogenase-elevating virus (LDV, strain P). Transcripts of open reading frames (ORFs) 2, 5, and 6 were in vitro translated in the absence and presence of microsomal membranes, and the products analyzed for molecular weight, sensitivity to endoglycosidase F/N-glycosidase F and proteinases, and reaction with anti-LDV antibodies. The ORF 6 mRNA translation was enhanced in the presence of microsomal membranes. ORF 6 encodes a polytopic class III membrane protein identified as the nonglycosylated virion envelope protein (M/VP-2; approximately 18 kDa). The protein has a very short (about 11 amino acids) ectodomain, a longer (about 79 amino acids) C-terminal endodomain, and crosses the membrane three times between these domains. ORF 5 encodes the primary virion envelope glycoprotein (VP-3P) (25-42 kDa). Our results suggest that it is a polytopic class I glycoprotein. After removal of a signal peptide, the processed protein of about 171 amino acids consists of a short (approximately 30 amino acids) N-terminal ectodomain with three asparagine residues that appear to be N-glycosylated, a segment that crosses the membrane three times, and an about 74 amino acid long C-terminal endodomain. Neutralizing anti-LDV antibodies are probably directed to an epitope(s) in the N-terminal ectodomain. The ORF 2 protein is a standard class I glycoprotein with a single C-terminal membrane anchor segment and its signal peptide is removed during membrane-associated synthesis. The remaining ectodomain (about 165 amino acids) contains three asparagine residues which appear to be N-glycosylated. Our results suggest that the ORF 2 protein may be present in a low concentration in LDV virions (VP-3M).

Lactate dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNA leader and comparison of 3'-terminal sequences of two LDV isolates.

The 3'-terminal 1314 nucleotides of the genome of one isolate of lactate dehydrogenase-elevating virus, LDV-P, has been derived by sequence analyses of cDNAs from several genomic libraries and compared to that of another LDV isolate, LDV-C (Godeny et al. (1990) Virol. 177, 768-771). The 3'-non-coding segment of 80 nucleotides of the two LDV genomes is identical, whereas marked, but varying nucleotide and amino acid divergence is apparent in the three upstream overlapping open reading frames (ORF). The third ORF from the 3'-end exhibits only 82% nucleotide and 90% amino acid identity, whereas the 3'-terminal ORF, which encodes the nucleocapsid protein, exhibits approximately 99% amino acid identity. The second 3'-terminal ORF encodes an 18.8 kDa protein which lacks N-glycosylation sites but possesses 2 or 3 potential transmembrane helices in the N-terminal half of the molecule. A similar membrane organization is observed for the corresponding protein of equine arteritis virus and the M protein of mouse hepatitis virus. The sequence analyses combined with Northern hybridization analyses of RNA from LDV-infected macrophages and spleens of LDV-infected mice indicate that the three ORFs encoded by the 3'-terminal end of the LDV genome are expressed via the three smallest mRNAs (mRNAs 6-8) of the seven subgenomic mRNAs of LDV (mRNAs 2-8), which range in size from about 0.8 to 3.6 kb. All mRNAs have been shown to carry poly(A)-tracts and a common leader sequence. The seven mRNAs were produced in infected macrophage cultures concomitantly with genomic LDV RNA. Maximum LDV RNA synthesis was observed between 6 and 8 h post-infection. The same seven subgenomic mRNAs were detected in macrophages infected with three different isolates of LDV, but different relative amounts of some of the mRNAs were produced. The relative proportions of molecules of mRNAs 1-8 present in 6 h LDV-P-infected macrophages were about 13, 5, 5, 8, 6, 11, 11 and 27% of the total, respectively.