No abstract

Avian reovirus S1133 specifies at least 10 primary translation products, eight of which are present in the viral particle and two of which are nonstructural proteins. In the work presented here, we studied the covalent modifications undergone by these translation products in the infected cell. The structural polypeptide 2 was shown to be intracellularly modified by both myristoylation and proteolysis. The site-specific cleavage of 2 yielded a large carboxy-terminal fragment and a myristoylated ϳ5,500-M r peptide corresponding to the amino terminus. Both 2 and its cleavage products were found to be structural components of the reovirion. Most avian reovirus proteins were found to be glycosylated and to have a blocking group at the amino terminus. In contrast to the mammalian reovirus system, none of the avian reovirus polypeptides was found to incorporate phosphorus during infection. Our results add to current understanding of the similarities and differences between avian and mammalian reoviruses.Avian reoviruses are very similar in structure and molecular composition to mammalian reoviruses. Both groups have a genome consisting of 10 segments of double-stranded RNA surrounded by a two-layer capsid which also encloses a number of short oligonucleotides (13). However, avian reoviruses differ from their mammalian counterparts in lacking hemagglutinating activity (11) and in being able to induce fusion of cultured cells (27).Mammalian reovirus type 3, the prototype of the genus Orthoreovirus, encodes 11 primary translation products which can be separated into three size classes: large (), medium (), and small () (38). The viral polypeptide 1, encoded by the M2 genomic segment, is proteolytically cleaved in the infected cell to yield 1C and 1N, the latter being a small peptide that corresponds to the amino terminus of the precursor. All three polypeptides are structural components of the virion (30). Intracellular covalent modifications of mammalian reovirus proteins have also been documented. Specifically, 1 and 1N contain an amide-linked myristoyl group attached to their amino-terminal glycines (30), and the presence of this group is necessary for the subsequent site-specific cleavage of 1 to 1C in transfected COS cells (43). There is also evidence for the presence of phosphoserine residues in 1C (16), and both 1 and 1C have been shown to be polyadenylylated and/or ADP-ribosylated (3,4). The presence of glycoproteins in mammalian reovirions was first suggested by the fact that treatment of purified virions with ␤-glycosidase, potassium periodate, sodium borohydride, or lysozyme reduced both the hemagglutination titer and the infectivity of the virus (19,20,42). However, subsequent work on the glycosylation of mammalian reovirus proteins has produced conflicting results: Krystal et al. reported (15) that 1C was the only polypeptide glycosylated, whereas the results of Lee (18) suggested that all mammalian reovirus polypeptides except 2 are glycoproteins. As far as we are aware, there have been no subsequent attempts...

Previous reports have suggested that avian reovirus S1133 fails to replicate in mouse L cells. In this article, we report that replication does occur under certain culture conditions. The avian reovirus was found to grow in mouse L cells at pH 6.4 and 7.2 but not at pH 8.2. Culture medium with a basic pH directly inhibited viral transcription and genome replication. As a result, viral protein synthesis was also affected. At permissive pH levels, avian reovirus grew better in monolayers than in suspension cultures of L cells because of the influence of cell attachment status on viral macromolecular synthesis. Our results not only show that avian reovirus can replicate in mouse L cells but also help to explain why it did not in previous studies.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.