Precise Mapping of the Replication and Transcription Promoters of Human Parainfluenza Virus Type 3

Abstract

The terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for directing RNA replication and transcription. The 3' terminus (leader region) of the negative-sense, genomic RNA of the rhabdoviruses and paramyxoviruses is known as the leader (Le) promoter and directs synthesis of positive-sense replication and transcription products. The 3' terminus of the antigenome is termed the trailer complementary (TrC) promoter and directs the synthesis of genomic RNA. By creating mutations in the corresponding regions of an HPIV3 minireplicon in which the viral protein coding sequences were replaced by the luciferase gene, we were able to precisely define the elements of the leader promoter involved in directing positive-strand replication of HPIV3. Nucleotides 1 through 12 (from the terminus) formed a domain critical for replication. The region from nucleotides 13 through 55 was important but not crucial for replication, while G residues at positions 79, 85, and 91 comprised another domain critical for replication. It was also shown that the TrC promoter is similar, though not identical, to the Le promoter. Nucleotides 1 through 12 of the TrC promoter were critical for synthesis of genomic RNA, though specific positions behaved differently from the corresponding positions of the Le promoter. While many of these mutations could not be analyzed for transcription because they completely abrogated genomic RNA synthesis (the template for transcription), we were surprised to find that no mutations in the leader promoter which decreased replication had any significant effect on transcription. However, mutations in the intergenic sequence and gene start signal following the leader and preceding the luciferase message severely decreased transcription, but not replication.

Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity

“…Seven of the eight residues contained in nt 36 to 43 of the RSV Le are U, suggesting that this U-rich nature is an important feature. The finding that nucleotides at the end of Le are important for transcription is consistent with findings for VSV, SeV, and parainfluenza virus type 3 (4,20,27,39), suggesting that the sequences immediately upstream of the promoter-proximal GS signal are important for many mononegaviruses. However, it should be emphasized that although nt 36 to 43 increased the efficiency of initiation from the GS signal, this region was not required for correct initiation, synthesis of complete mRNA, or the initiation of stop-start sequential transcription.…”

“…In the case of VSV, it has been shown that a synthetic template containing 17 nt of wt sequence is a functional promoter in vitro (34), and the first 15 nt of wt sequence can act as a promoter in an intracellular minigenome assay (39). The first 12 nt of the genome and antigenome are conserved in all of the Paramyxovirinae, and these nucleotides have been shown to be critical for RNA replication in parainfluenza virus type 3 (20), suggesting that this is the typical size of the 3Ј-terminal element of the mononegavirus replication promoter. In the case of members of the Paramyxovirinae, the replication promoter contains a second essential cis-acting element located within the first gene (20,21,29,37), but this additional element is not present in RSV or VSV.…”

“…Initiation of replication and transcription occurs at or near the 3Ј end of the genome (11) and is directed by cis-acting sequences located within a 3Ј extragenic region known as the leader (Le) (4,14,15,20,27,39). For members of the heterologous Paramyxovirinae subfamily of Paramyxoviridae, the genome promoter is more complex and extends into the first gene (20,21,29,37). During transcription, the polymerase responds to sequences at the boundaries of each gene to generate monocistronic mRNAs (1,23,35).…”

Role for the Paramyxovirus Genomic Promoter in Limiting Host Cell Antiviral Responses and Cell Killing

“…For many paramyxoviruses, such as SV5, SeV, and human parainfluenza virus type 3, the genomic promoter contains two 18-base promoter elements (PrE) that are essential for viral RNA synthesis (19,43,31,32). PrE-I is located at the ultimate 3Ј end of the genome and is separated from a second internal PrE, PrE-II, by a sequence-independent spacer region ( Fig.…”

Inhibition of human parainfluenza virus type 3 infection by novel small molecules

“…In vivo HPIV3 minigenome assay was performed as described previously (Hoffman et al, 2000) with slight modifications. HeLa cell monolayers in 12 well plate, grown to 90% confluency, were infected with recombinant vaccinia virus vTF7-3, which expresses T7 RNA polymerase, at an MOI of 3.…”