Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein 1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound 1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of 1 as a determinant of thermostability. Intact virions, which contain 3 bound to 1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for 3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.The virions of mammalian orthoreoviruses (reoviruses) contain viral proteins arranged in two concentric icosahedral layers, commonly called the outer and inner capsids. During treatments with exogenous proteases in vitro, three proteins from the outer capsid can be sequentially removed to yield two well-characterized disassembly intermediates: the infectious subvirion particle (ISVP) and the core. ISVPs differ from virions in having lost the major outer-capsid protein 3. In addition, the other major outer-capsid protein, 1, which appears to have been cleaved near its N terminus in virions to yield particle-bound fragments 1N and 1C (29), has been cleaved again near its C terminus in ISVPs to yield the additional particle-bound fragments 1␦/␦ and (26). Cores differ from virions in having lost not only 3 but also 1 and its fragments as well as the receptor-binding outer-capsid protein 1. Studies of these subvirion particles have been crucial for localizing proteins within the outer capsid as viewed by cryoelectron microscopy and three-dimensional image reconstruction (14).In addition to their uses for studies of reovirus structure, ISVPs and cores are thought to represent disassembly intermediates that play essential roles during productive infection. Cores are active at transcription in vitro and according to one hypothesis represent the primary transcriptase particles that gain access to the cytoplasm during entry into cells and first synthesize the viral plus-strand RNAs for translation and packaging (6). Cores are poorly infectious through binding and uptake from the cell surface, s...

Reovirus nonstructural protein NS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of NS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of NS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The NS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, NS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, NS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl 2 , distinguishing it from a classical ATP-dependent helicase. These properties of NS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for NS in replication of the reovirus RNA genome.Mammalian orthoreoviruses (reoviruses) encode three nonstructural proteins, NS, NS, and 1s, whose roles during viral infection remain poorly understood. This report concerns NS, which is known to be essential for reovirus replication based on the phenotype of a conditionally lethal (temperaturesensitive) mutant with its lesion in the NS-encoding S3 gene segment (9, 27). The NS protein comprises 366 amino acids and has a molecular mass of 41 kDa. Interaction of NS with the viral plus-strand RNAs in infected cells is well documented (3,14). Moreover, when NS from infected cells is used to bind those RNAs in vitro, it protects 20-to 40-nucleotide fragments of the RNAs from RNase T1 digestion (34). Since the protected fragments include the 3Ј ends of at least some of the plus-strand RNAs, it was proposed that NS binds specifically to those regions (34). In addition, NS and two other reovirus proteins, NS and 3, are found to associate with the viral plus-strand RNAs shortly after they are transcribed in infected cells and before minus-strand synthesis converts them into the double-stranded RNA (dsRNA) genome segments (3). These findings have led to a hypothesis that NS plays a role in selecting or condensing the viral plus-strand RNAs for packaging during early stages of particle morphogenesis (3,14,28,34). A role for NS in translation of proteins from the reovirus plus-strand RNAs has also been suggested (10,14).Evidence for a direct role of NS in minus-strand synthesis is limited. The temperature-sensitive mutant whose lesion maps to the S3 gene segment (27) produces little or no dsRNA at restrictive temperatures (9, 15), but this indicates only that NS provides a required function at or before minus-strand synthesis in the replication cycl...

No abstract

The early growth response 2 (Egr2/Krox-20) transcription factor is essential for myelination of the peripheral nervous system and segmentation of the vertebrate hindbrain. To probe the mechanism by which Egr2 is regulated, we used a yeast two-hybrid assay and identified an RNA helicase, Ddx20 (DP103/Gemin3), as an Egr2-interacting protein. Mammalian two-hybrid assays indicated that Ddx20 can interact with Egr1, Egr3, and Egr4, in addition to Egr2, making it the only known cofactor that interacts with all four Egr family members. Using several Egr2 target promoters, we found that Ddx20 repressed Egr2-mediated transcriptional activation with significant promoter specificity. In addition, Ddx20 repressed Egr2-mediated activation of the endogenous insulin-like growth factor 2 (IGF2) gene. Interestingly, the C-terminal segment of Ddx20, which lacks the DEAD box helicase domain, was sufficient for its robust and specific repression. We also examined possible interactions between Ddx20 and Nab proteins, the only other known corepressors of the Egr family, and found that these two corepressors act independently. Finally, transcriptional repression assays performed in the presence of a histone deacetylase inhibitor (trichostatin A) indicate that although repression of certain promoters by Ddx20 requires histone deacetylase activity, another repression mechanism must also be involved. Because Egr2 is critical for hindbrain development and peripheral nerve myelination, modulation of Egr2 by Ddx20 may play an important role in maintaining the correct expression level of Egr2 target genes.The members of the early growth response (Egr) 1 family of transcription factors are rapidly induced by various extracellular signals such as growth factors and hormones, as well as developmental and environmental signals. Once induced, Egr proteins activate genes that cause cellular responses such as proliferation, differentiation, or apoptosis depending upon the stimulus. The Egr proteins share a highly conserved DNAbinding domain consisting of three C 2 H 2 zinc fingers that recognize a GC-rich sequence, as well as shorter regions of homology outside of the DNA-binding domain (1, 2). Although all four Egr family members share a similar DNA-binding specificity (3), targeted disruption of these genes demonstrates distinct physiological roles for these proteins (4). One of the most dramatic phenotypes in this family arises from targeted disruption of Egr2/Krox-20 (hereafter referred to as Egr2); homozygous null mutants of Egr2 all die within 1 or 2 weeks after birth and exhibit defects in hindbrain patterning, peripheral nerve myelination, and bone formation (5-8).A series of elegant experiments have described the role of Egr2 in establishing the segmentation pattern of the developing vertebrate hindbrain. Targeted disruption of Egr2 causes the disappearance of rhombomeres r3 and r5, hindbrain segments in which Egr2 is normally expressed. Egr2 regulates several homeobox genes (Hoxa2, Hoxb2, and Hoxb3) (9 -11), which help determine the anter...