Mutational analyses of the nonconserved sequences in the Bunyamwera Orthobunyavirus S segment untranslated regions - PubMed (original) (raw)
Comparative Study
Mutational analyses of the nonconserved sequences in the Bunyamwera Orthobunyavirus S segment untranslated regions
Anice C Lowen et al. J Virol. 2005 Oct.
Abstract
Bunyamwera virus (BUNV) is the prototype of the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite genome of negative-sense RNA composed of small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments and throughout the genus, while the internal regions are unique to each segment and largely nonconserved between different viruses. To investigate the functions of the UTR sequences, we constructed a series of BUNV S segment cDNA clones with deletions in the 3' and/or 5' UTR and then attempted to rescue these segments into recombinant viruses. We found that the genomic 5' UTR was much more sensitive to mutation than the 3' UTR and, in general, sequences proximal to the termini were more important than those flanking the coding region. Northern blot analyses of infected-cell RNA showed that the internal, nonconserved sequences of the S segment 3' UTR play a role in the regulation of transcription and replication and the balance between these two processes. In contrast, deletions in the 5' UTR caused attenuation of the recombinant virus but did not specifically affect levels of S segment RNAs or the encoded nucleocapsid protein. Thus, the internal regions of both UTRs are functional: most of the 5' UTR is essential to viral growth, and, while nonessential, the internal 3' UTR is important to the regulation of viral RNA synthesis.
Figures
FIG. 1.
Sequence alignment of the 3′ (A) and 5′ (B) S segment untranslated regions of nine members of the Orthobunyavirus genus. Red denotes identity in 80% of sequences; blue denotes conservation of a purine or pyrimidine in 80% of sequences. Abbreviations: INKV, Inkoo virus; JCV, Jamestown Canyon virus; LACV, La Crosse virus; TAHV, Tahyna virus; AKAV, Akabane virus; TINV, Tinaroo virus; MAGV, Maguari virus.
FIG. 2.
Rescue of recombinant viruses containing deletions in the S RNA segment. The upper part shows a schematic diagram of recombinant S genome segments. Solid black bars represent untranslated regions; patterned bars represent the N open reading frame; dotted lines indicate deleted regions. The name of each recombinant segment follows the format Sx/y, where x is the length of the 3′ UTR and y is the length of the 5′ UTR, in nucleotides. + denotes recovery of a recombinant virus; − denotes no recovery. The lower part shows plaque phenotypes of wild-type BUNV and recombinant viruses on Vero-E6 cells. Cell monolayers were fixed 5 days postinfection with 4% formaldehyde and stained with Giemsa solution.
FIG. 3.
Growth of wild-type BUNV and UTR deletion mutants in cell culture. BHK-21 cells were infected at a multiplicity of infection of 1, and virus released into the growth medium was titrated by plaque assay at the time points indicated.
FIG. 4.
Analysis of viral RNAs from BHK-21 cells infected with wild-type and recombinant viruses. (A) Northern blot using probes complementary to S and M positive-sense RNAs. The bands seen in the middle of the blots represent nondenatured S RNA dimers. (B) Ratio of quantified S mRNA to antigenome RNA levels for wild-type BUNV and the 3′ UTR deletion mutants. (C) Northern blot using probes complementary to S and M genomic RNAs. (D) Levels of S genome segment in each infection, normalized to the corresponding M segment levels.
FIG. 5.
Protein synthesis in BHK-21 cells infected with wild-type BUNV and recombinant viruses. (A) In vivo protein labeling. Cells were infected at 1 PFU/cell and labeling with [35S]methionine was carried out 24 h postinfection, for 2 h. One-half the volume of lysate used for infected samples was loaded into the mock lane. Positions of viral proteins are indicated at the left. (B) Western blot of S segment-encoded proteins from wild-type and mutant virus-infected cells. The top panel shows N protein, and the two lower panels show NSs protein. The bottom panel is a longer exposure included to show that NSs is expressed by all of the viruses. Letters below the blots denote the nucleotide (nt) present at the −3 position relative to the N start codon in each S segment.
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