Jean L. Patterson

Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells

We examined the antigenic reactivities and virion associations of glycoproteins that were released into the culture fluids of cells infected with respiratory syncytial (RS) virus. Culture fluids and cell extracts were obtained from cells 24 to 30 h after they were infected with the Long strain of RS virus. Radioimmune precipitation of [3H]glucosamine-labelled glycoproteins by large glycoprotein (G)-specific or fusion protein (F)-specific monoclonal antibodies (MAbs) revealed that the G, F1 and F2 proteins were present in cell extracts but only the G protein was clearly evident in culture fluids. A glycoprotein (Mr 43K) which may be a precursor or a breakdown product of the G protein was also precipitated by the G-specific MAb from cell extracts and culture fluids. The G protein in culture fluids was slightly smaller (Mr 82K) than the G protein in cell extracts (Mr 88K). An abundant or heavily labelled, 18K glycoprotein in the fluids of virus-infected but not of mock-infected cells was weakly precipitated by the F-specific MAb; this suggested that the 18K protein shares epitopes with the fusion protein of RS virus. The absence of F1 and F2 polypeptides from culture fluids is evidence that the cells, which contained an abundance of these proteins, were intact. To determine whether any of the viral glycoproteins released by infected cells were soluble (non-virion-associated), culture fluid was subjected to rate zonal centrifugation in a 10 to 50% sucrose gradient. An assay of fractions using a MAb-capture ELISA for the nucleocapsid (N) and F proteins revealed a peak of activity, due to virions, in the centre of the gradient, and a strong signal for the N protein at the top of the gradient suggesting that N protein was released from intact cells. Radioimmune precipitation of glycoproteins from the fractions at the top of the gradient using a hyperimmune guinea-pig serum revealed the G protein and a heterogeneous band which had the electrophoretic mobility of the 43K protein. Neither the F1 nor the F2 protein was present in these fractions thus suggesting that virions had remained intact. These results showed that a soluble form of the G protein of RS virus is released into the culture fluids of intact, infected cells. Several theories concerning viral and non-viral origins for the 18K protein are discussed.

Effect of phosphorylated ribavirin on vesicular stomatitis virus transcription.

The effect of phosphorylated ribavirin on a vesicular stomatitis virus (VSV) in vitro transcription reaction was examined. Viral mRNA synthesized in the presence of the 5 mono-, di-, and triphosphorylated forms of the drug translated with equal efficiencies under the test conditions. However, all three phosphorylated species inhibited VSV transcription. The mono- and diphosphorylated forms of the drug possessed approximately two to three times the inhibitory activity as the triphosphorylated form. Transcripts synthesized in the presence of drug were full length and were absent of incorporated drug. Inhibition by ribavirin 5-diphosphate could be reversed by the addition of UTP, CTP, and GTP, while the addition of GDP to the reaction did not reverse inhibition. Ribavirin diphosphate was added to a La Crosse virus in vitro transcription assay to determine whether an inhibitory effect could be established in a viral system that was more sensitive to ribavirin than was VSV; it led to profound inhibition of RNA synthesis at concentrations as low as 0.1 microgram/ml. These data suggest that ribavirin has an effect on the initial steps of transcription by some RNA-dependent RNA polymerases and that this effect may be mediated by several phosphorylated forms. Images

Molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction.

The effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro transcription reaction was examined. Analysis of the kinetics observed when the concentrations of nucleoside triphosphates were varied was performed with vesicular stomatitis virus wild-type standard virions. Double-reciprocal and Eadie-Hofstee plots showed competitive inhibition with all natural nucleoside triphosphates when both ribavirin diphosphate (RDP) and ribavirin triphosphate (RTP) were used. The Km values for ATP obtained for the wild-type polymerase were similar to those reported previously. To further characterize the observed inhibition kinetics, in vitro transcription products synthesized in the presence or absence of RDP and RTP were purified by CsCl centrifugation and were primer extended with oligonucleotides specific for either positive-sense leader or nucleocapsid mRNA transcripts. The ratios of leader to nucleocapsid mRNA were measured from primer-extended in vitro transcription products. It was found that the addition of RDP or RTP did not significantly change the in vitro ratio, suggesting that the polymerase is blocked before it enters the 3 end of the template. Images

Cultivation of Leishmania braziliensis in an economical serum-free medium containing human urine

Leishmania braziliensis cells are difficult to culture in vitro and usually require media supplemented with serum for sustained cell division. Fresh, sterile urine is an inexpensive substitute for serum in the culture of 2 strains of L. braziliensis, 1 infected with Leishmania RNA virus 1, and 1 uninfected. In the presence of urine, both the infected and the uninfected strains grew to the same final cell density as the same strains grown in the presence of serum. One strain of Leishmania major was also successfully cultured in urine-supplemented media.

Transcribing and replicating particles in a double-stranded RNA virus from Leishmania

During the replicative cycle of many double-stranded RNA viruses, transcription of particles with a double-stranded RNA genome alternates with replication of particles containing a single-stranded genome. In virions infecting some strains of Leishmania guyanensis the putative transcriptase and replicase activities of the RNA-dependent RNA polymerase were previously detected in vitro. Northern hybridization to RNA of known polarity demonstrates that the single-stranded RNA products are of positive polarity and, by definition, are the products of the viral transcriptase. Re-evaluation of previously published data in the light of these findings suggests that transcription in Leishmania viruses is conservative. Sedimentation in sucrose gradients revealed two types of viral particles; single-stranded RNA particles comprised a small fraction of the virus population and sedimented more slowly than the peak of double-stranded RNA particles. In agreement with the replicative model of other dsRNA viruses, these single-stranded particles co-purified with the viral replicase activity that resulted in double-stranded RNA synthesis. In virus-infected promastigote extracts replicase activity decreased with increasing parasite density in culture, suggesting a correlation between cell division and viral replication.

Altered ATP function of a vesicular stomatitis virus mutant detected by kinetic analysis of the transcriptase using phosphorylated ribavirin

We have studied the effect of phosphorylated ribavirin on the vesicular stomatitis virus (VSV) in vitro polymerase reaction by analysis of kinetic data obtained by varying the concentration of nucleoside triphosphates. The wild-type VSV had previously shown a competitive inhibition with the four natural nucleoside triphosphates with the use of ribavirin diphosphate (RDP) or ribavirin triphosphate (RTP). In contrast, when RDP (or RTP) was added to a transcription assay system using the polR1 mutant of VSV, a non-competitive or mixed type of inhibition was observed when the concentration of ATP was varied. Our results indicate that polR1 has an altered ATP function in addition to the previously described phenotypic characteristics of this mutant, which include synthesis of readthrough products of the leader/nucleocapsid (N) gene junction and a decreased ATP requirement for transcription. We have also studied CsCl-purified in vitro transcription products by primer-extending leader or N mRNA transcripts and found that the ratio of leader/N mRNA for VSV polR1 (1.3:1) was lower than values obtained previously for wild-type (3.7:1).

Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus

A soluble form of the G glycoprotein, the attachment protein, of respiratory syncytial virus is shed from infected HEp-2 cells. The Gs proteins of the Long and 18537 strains have apparent molecular sizes of 82 and 71 kilodaltons, respectively, 6 to 9 kilodaltons smaller than the virion-associated forms (Gv). The Gs protein of the Long strain was further characterized. Approximately one in six of all of the radiolabeled G molecules in these cultures at 24 h postinfection was present as the Gs protein. The Gs protein was clearly evident in culture fluids at 6 h postinfection, but the Gv protein could not be discerned until 12 h after infection, an observation that is consistent with the 12-h eclipse period for respiratory syncytial virus. Therefore, the Gs protein is shed, in part at least, from intact, infected cells and before the appearance of progeny virus. The appearance of a smaller Gs protein (74 kilodaltons) in fluids of infected calls which were incubated with tunicamycin shows that addition of N-linked oligosaccharides is not required for the genesis and shedding of the Gs protein. Sequencing of the amino terminus of purified Gs protein revealed two different termini, whose generations are consistent with cleavages of the full-length G protein between amino acids 65 and 66 and between residues 74 and 75. This result suggests that the Gs protein is present in two different forms which lack the proposed intracytoplasmic and transmembrane domains of the full-length G protein.

EFFECT OF RIBAVIRIN ON VIRAL TRANSCRIPTION

We have examined the effect of ribavirin on vesicular stomatitis virus (VSV) RNA synthesis using a cell-free transcription system. Our prior data indicated that ribavirin had little effect on primary transcription of VSV mRNAs synthesized intracellularly, but that these RNAs were translated inefficiently. Consistent with these data, VSV cell-free transcription was inhibited only mildly (25%) by ribavirin triphosphate at 100 mg/ml. However, ribavirin diphosphate (RDF) and, to a lesser extent, ribavirin monophosphate, were 2-3 times as inhibitory at the same concentration. Transcripts synthesized in the presence of RDP were full lengthed. The inhibition by RDP could be reversed by increasing the concentration of GTP. However, reversal could also be achieved by increasing the concentration of UTP, CTP, and ATP, but not by the addition of GDP. RDP was added to a LaCrosse virus cell-free transcription assay to determine if an inhibitory effect could be established in a viral system more sensitive to ribavirin than VSV; addition of RDP to this reaction led to profound inhibition of RNA synthesis at concentrations as low as 0.1 ug/ml. These data suggest that in some viruses ribavirin may have a major direct effect on initial steps of transcription and that this effect may be mediated by the di- and monophosphorylated forms.

Transcription by La Crosse Virus

The genome of La Crosse (LAC) virus, a member of the California encephalitis serogroup of the insect-transmitted Bunayviridae (1), consists of 3 segments of sing le-stranded RNA of negative polarity (-) each contained within a separate nucleocapsid labelled small (S), medium (M), and large (L). Genetic and molecular studies have led to the following gene assignments: the S segment codes for the N protein, the M segment codes for the two surface glycoproteins, and the L segment, by elimination, codes for the L protein which is located internally and thought to be part of the viral polymerase (2,3,4). In addition to the structural proteins, bunyavirus-infected cells also contain at least two nonstructural (NS) proteins, NSs and NSm, coded for by the S and M genome segments respectively (5). The complete nucleotide sequences of the S genome of LAC and snowshoe hare (SSH) viruses have recently been determined (6,7). In both viruses, the S segment has been shown to contain two overlapping open reading frames (ORF).