Accumulation of Terminally Deleted RNAs May Play a Role in Seoul Virus Persistence

Abstract

Two independent, long-term infections were analyzed to determine whether changes in viral replication could contribute to the establishment and/or maintenance of persistent Seoul virus infections. Infected cell cultures initially contained high levels of infectious virus and intracellular viral RNA that peaked between approximately 7 to 16 days postinfection and then gradually declined until day 26. After day 26, the viral titers and the levels of the small (S), medium (M), and large (L) viral RNAs varied cyclically until the end of the studies. The changes in the concentrations of the RNAs and titer were similar in pattern and appeared to result from changes in the regulation of replication. Neither internal deletions nor an accumulation of nucleotide changes were found in the RNAs. However, fine mapping and sequence analysis revealed short deletions in some of the RNAs in the conserved complementary terminal sequences believed to contain the signals for initiation of replication and transcription. Deletions at the 3 termini of S, M, and L virus-sense RNAs (vRNAs) accumulated during the acute phase of infection just before the time that the viral titer and the concentration of vRNAs and virus complementary-sense RNAs (cRNAs) began to decline. The absence of deletions at the 5 termini of the S, M, and L cRNAs suggests that the 3-deleted vRNAs may not be replication competent. Thus, as the percentage of 3-deleted vRNAs increase in the population, they could potentially compete with standard virus and downregulate viral replication. Deletions at the 3 L cRNA and 5 L vRNA termini were also observed, and the proportion of these deleted RNAs varied cyclically during the infections. We propose a model in which terminal nucleotide deletions arise by nuclease activity of the viral polymerase. In addition, we speculate that cleaved terminal fragments might be used as primers during replication, resulting in the repair of some of the deleted RNAs.The Hantavirus genus, in the family Bunyaviridae, is composed of a large group of enveloped negative-strand RNA viruses with a tripartite segmented genome (32). The three segments, small (S), medium (M), and large (L), encode the viral nucleocapsid (N), envelope glycoproteins (G1 and G2), and polymerase (L), respectively (33,35,36). Hantaviruses replicate in the cell cytoplasm, and replication is likely to proceed by mechanisms similar to those used by other negative-strand RNA viruses. That is, synthesis of mRNA and antigenome or virus complementary-sense RNA (cRNA) is initiated from the 3Ј terminus of viral RNA templates, and the synthesis of virus-sense RNA (vRNA) is initiated from the 3Ј terminus of cRNA templates. The signals for initiation of replication and transcription have not yet been defined but are believed to reside in the imperfect inverted repeat present at the termini of each genomic segment. This sequence is approximately 20 nucleotides long and is conserved among all members of the genus. Because bunyavirus ribonucleocapsids are circular (24, 26) and the termin...

Genome trimming: A unique strategy for replication control employed by Borna disease virus

“…6). In Seoul virus, another member of the Hantavirus genus, the 3Ј and 5Ј termini of vRNA and cRNA exhibit terminal deletions if the virus is derived from persistently infected cells (29). It was speculated that accumulation of terminally deleted RNAs may play a role in Seoul virus persistence.…”

Persistent hantavirus infections: characteristics and mechanisms

“…The length and concentration of the L RNA deletions were found to vary during persistence and correlate with reduced levels of viral replication. We postulate that these terminally deleted RNAs have a causal role in downregulating replication and viral gene expression 43 . A model has been proposed in which terminal nucleotide deletions arise via the nuclease activity of the viral polymerase.…”

“…2). Although not yet defined, it is possible that both the terminal nucleotide sequence and the panhandle structure are important signals for correct transcription initiation of the viral RNAs and the viral-complementary RNAs. To investigate this possibility, changes in the termini and other regions of the S, M and L RNAs that arose during establishment and maintenance of persistence were characterized from two independently derived Seoul virus infections of cultured mammalian cells, one lasting 46 days and the other lasting 139 days 43 . The cultured cells mimicked the early stages of rodent infection in that they had an acute, non-cytolytic stage with peak virus titers one to two weeks after infection.…”

“…2. The 3Ј and 5Ј terminal nucleotides of the L, M and S genomic segments of Seoul virus and their predicted base pairing to form panhandle structures [43][44][45] .…”

How RNA viruses maintain their genome integrity

“…Evidence for additional non-templated residues has been found for the positive-strand RNA viruses cucumber mosaic virus (Burgyan & Garcia-Arenal, 1998) and dengue virus (Teramoto et al, 2008), and the negative-strand RNA viruses Borna disease virus (BDV), lymphocytic choriomeningitis virus (LCMV) and Hantaan hantavirus (Meyer & Schmaljohn, 2000;Meyer & Southern, 1994;Schneider et al, 2005). Although the mechanism by which the additional nucleotides were added to these virus genomes is not known and could involve a terminal transferase activity, such as that described below, the random nature of the additional sequence is consistent with the non-templated initiation mechanism described for TCV.…”