- Volume 66, Issue 12, 1985

An antiserum made against a synthetic peptide from an internal region of the predicted amino acid sequence of the avian myeloblastosis virus (AMV) transforming v-myb(AMV) gene identified two products, p46v-myb(AMV) and p32v-myb(AMV), which were localized in the nucleus of AMV-transformed myeloblasts. We propose that these proteins are the in vivo products of the v-myb(AMV) gene and thus the transforming protein(s) of AMV.

A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to (i) vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and (ii) using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.

Evidence is presented for the production in dengue type 2 virus (DEN-2)-infected BHK cells of large virus-specific proteins with molecular weights up to 250000. These proteins were most prominent in lysates of cells which had been labelled with [35S]methionine for 7 to 15 min. During pulse-chase experiments, these high mol. wt. proteins appeared to be converted into smaller, more stable, proteins with mol. wt. between 10000 and 98000. Finally, inhibition of proteolysis prevented the chasing of label from the high mol. wt. proteins to the smaller viral proteins which normally accumulate in DEN-2-infected cells. These findings are consistent with the idea that processing of large polyprotein precursors plays an important role in the production of flavivirus proteins.

Mammalian and arthropod cell cultures were used to assess the neutralizing activity of six monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus. Four antibodies, two neutralizing and two non-neutralizing, showed no host-dependent differences, giving similar results when post-treatment infectivity was determined using either Aedes albopictus cells or BHK-21 cells. For two other antibodies, however, dissimilar activities were observed between the vertebrate and invertebrate cell lines. One of these antibodies was positive when BHK-21 cells were employed as the post-treatment host and negative when mosquito cells were used; the other antibody was the converse. The epitope for this last antibody was present on all California serogroup viruses examined, which suggests that it may have a special significance in the natural life-cycle of the virus.

The effect of interferon (IFN) on infection and maintenance of persistent infection of Borna disease (BD) virus in cell cultures was investigated. Acutely BD virus-infected primary rabbit brain and rat lung cells produced significant levels of interferon detectable 3 days post-infection in the culture supernatants. Rat brain and rat lung cells persistently infected with BD virus produced only moderate levels of IFN over a long period. In contrast, persistently infected Madin-Darby canine kidney (MDCK) cells did not produce detectable amounts of IFN. Exogenous homologous IFN completely inhibited the expression of BD virus antigen in acutely infected rabbit brain cells, when added during the first 24 h after infection. IFN added later (2 to 6 days post-infection) reduced virus titres to different degrees depending on the onset of treatment. However, IFN added to persistently infected rat lung cells did not appear to influence the degree or quality of BD virus antigen expression or the intracellular amount of infectious virus. Two facts indicate that IFN is not involved in the establishment or maintenance of persistent BD virus infection in vitro. Thus, MDCK cells, which could not be induced to produce IFN, can be readily persistently infected with BD virus in vitro, and exogenous IFN did not appear to influence persistent BD virus infection.

Components of herpes simplex virus remained bound to the diploid cell membrane after nucleocapsid penetration into the cytosol. These components enabled the infected cells to induce interferon-alpha (IFN-α) in peripheral blood mononuclear cells even when the infected cells were fixed by glutaraldehyde. Monoclonal antibodies directed against the major viral glycoprotein D could neutralize their IFN-α-inducing capacity. Thus, the process of IFN induction does not require uptake and penetration of the inducer into the effector cells. The process was, however, sensitive to lysosomotropic drugs. These data suggest that a membrane receptor is involved in the IFN-α induction mechanism.

Isolates of bovine herpesvirus 1 (BHV-1) are associated with a variety of clinical manifestations. To determine if a single form of BHV-1 was responsible for the different virus-associated diseases or whether subpopulations of various isolates produced different clinical symptoms, studies were initiated to examine the DNA restriction enzyme patterns and nucleic acid homology between virus isolates from respiratory infections and other clinical syndromes. Differences between the genomes of several virus isolates were detected using DNA restriction enzyme analyses. However, nucleic acid hybridization studies of the virus DNAs using filter and liquid hybridization indicated at least a 95% genetic homology between the virus isolates from different types of infections. Additionally, these studies demonstrated that the DNA of BHV-1 had an average molecular weight of 84 × 106.

Rice gall dwarf virus (RGDV) was studied in infected rice plants and in its principal insect vector, the green leafhopper Nephotettix nigropictus, by electron microscopy. In plants, virions were restricted to the cytoplasm and vacuoles of phloem-related cells and of gall cells generated from the phloem. In the insects, virions occurred in the cytoplasm of many different kinds of cells including those of the salivary gland, the gut, the fat body, muscles and nerves. Virions in both plant and insect cells had dense cores 40 to 50 nm in diameter which were surrounded by a less dense region; the total diameter was 60 to 70 nm. Viroplasms, crystals and tubules were observed in cells both of plants and insects. Although virions often occupied most of infected plant cells, they occurred only in restricted areas of insect cells. The restriction of virions to phloemrelated cells in infected plants probably explains why yields of purified RGDV are usually about 1/30 of those of rice dwarf virus, which invades many types of cell. About 3% of the virus particles seen in thin sections of infected plants appeared to be empty. Empty particles were purified from infected rice plants and were found to lack nucleic acid and much of the 56000 mol. wt. protein present in intact particles.

Tobacco ringspot virus (TobRV) RNA was translated efficiently in rabbit reticulocyte lysate and directed the synthesis of two principal polypeptides, M r 207 × 103 (207K) and 116K, corresponding to the translation products of RNA-1 and RNA-2 respectively. In addition, a 112K RNA-2-encoded polypeptide was sometimes detected. The 116K polypeptide was immunoprecipitated with anti-TobRV serum, suggesting that it was a precursor to coat protein. When translations were performed in the presence of dithiothreitol, the 207K polyprotein was apparently cleaved to yield 37K and 180K polypeptides, with additional processing into 65K and 128K polypeptides. Cleavage of the RNA-2-encoded polyprotein also occurred, although to a much lesser extent than that of the 207K polyprotein; polypeptides of 88K and 54K, both immunoprecipitated with antiviral serum, were identified as RNA-2-encoded cleavage products.

In vitro translation of virion RNA from carnation mottle virus (CarMV) in a messenger RNA-dependent rabbit reticulocyte lysate (MDL) resulted in the synthesis of four virus-specific polypeptides of apparent M r 100000 (P100), 77000 (P77), 38000 (P38) and 30000 (P30). Partial peptide mapping experiments and in vitro translation in the presence of partially purified calf liver amber suppressor tRNA demonstrated that P30, P77 and P100 are a series of overlapping polypeptides generated by a double readthrough mechanism. In addition we report the infection of Chenopodium quinoa protoplasts with CarMV. The viral coat protein was detected in virus-infected protoplasts. Only one other infection-specific protein with an apparent M r of 100000 corresponded in size to any of the other in vitro translation products.