Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Abstract

The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5 and 3 ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A)⅐U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 ؎ 10°) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.T he influenza A virus, a member of the Orthomyxoviridae family, has a genome consisting of eight single-stranded RNA molecules of negative polarity. These eight RNA segments encode ten proteins, including three RNA-dependent RNA polymerase (RdRp) proteins (PA, PB1, PB2) and one nucleoprotein (NP). The transcription and replication of the viral genome are performed in the nucleus of infected cells by a ribonucleoprotein (RNP) complex that is composed of the three polymerase proteins, NP, and the viral RNA (vRNA). Replication of the vRNA produces a full-length copy of positive-sense RNA (cRNA). The cRNA is then used in the formation of another RNP complex, which serves to generate newly synthesized vRNA. Transcription is also performed by the same RNP complex; however, initiation occurs by the pirating of a 7-methyl guanosine cap structure from a host mRNA. Viral transcription produces an mRNA molecule that is 15 to 22 nucleotides (nt) shorter than cRNA and contains a poly(A) tail (1).The influenza A virus RdRp binds specifically to the partial duplex promoter (also referred to as the panhandle RNA), which is made from the 5Ј and 3Ј termini of each RNA genome segment. Within this partial duplex, 13 nt at the 5Ј end and 12 nt at the 3Ј end are highly conserved among most influenza A virus variants (Fig. 1A). All of the necessary signals for replication and genome packaging appear to reside in these terminal sequences (2), and several pieces of evidence imply a regulatory role for the terminal structure in viral transcription initiation (3-6), termination, and polyadenylation (7,8).The RdRps are for the most part virus-encoded polymerases that synthesize RNA from an RNA template without a DNA intermediate. Despite the unique features of RdRps, little has been deciphered about their mechanisms of promoter recognition and RNA synthesis. The promoter of the...

The influenza virus RNA synthesis machine

“…The structures of vRNA and cRNA promoter regions have been determined by NMR. [103][104][105] Although they show structural similarities, some marked differences are also apparent, that could account for their differential recognition by the virus polymerase. In fact, biochemical and genetic evidences suggest that distinct regions of the polymerase interact with vRNA or cRNA promoter regions.…”

Structure and Dynamics of Hemimethylated GATC Sites

“…Scalar 1 J CH and dipolar 1 D CH couplings were derived from natural abundance 1 H- 13 C HSQC experiments with and without Pf1 (ϳ10 mg/ml) at 30°C. Pf1 filamentous bacteriophage was purchased from ASLA, Ltd. Alignment tensor analysis of the observed residual dipolar couplings was performed as described previously (20). All structure calculations were performed by crystallography and NMR system (CNS) software (21).…”

Influenza A Virus Multiplication and the Cellular SUMOylation System

“…i) The structure of the viral promoters: Through the use of Nuclear Magnetic Resonance (NMR) and calculations for determining the 3D structure of the vRNA promoter, it has been shown that the vRNA promoter has a terminal stem, followed by an internal loop, and a proximal stem [81][82]. The terminal stem displays an inherent bend that allows the viral promoter to be easily melted, therefore possibly playing a regulatory role during the initiation of RNA synthesis [82].…”

“…The terminal stem displays an inherent bend that allows the viral promoter to be easily melted, therefore possibly playing a regulatory role during the initiation of RNA synthesis [82]. This intrinsic bending of the terminal stem might be of great relevance for transcription initiation, since the viral RdRp does not appear to have a helicase activity associated to it [82].…”

“…The terminal stem displays an inherent bend that allows the viral promoter to be easily melted, therefore possibly playing a regulatory role during the initiation of RNA synthesis [82]. This intrinsic bending of the terminal stem might be of great relevance for transcription initiation, since the viral RdRp does not appear to have a helicase activity associated to it [82]. Additionally, the nucleotides involved in the formation of the internal loop within the vRNA promoter correspond to the same nucleotides previously identified as the binding sites for the viral RdRp [82][83].…”