H1N1流感病毒聚合酶片段的CpG抑制及其对密码子偏爱性的影响

H1N1流感病毒的聚合酶具有RNA复制、转录等功能,并且对流感病毒片段包装、子代繁殖及宿主适应性等有着重要作用。通过分析人、猪及禽类H1N1流感病毒聚合酶片段的二核苷酸频率及同义密码子的偏爱性,发现不同宿主中,流感病毒聚合酶片段的CpG频率最低,且均被强烈抑制;通过三类宿主间的比较发现,人流感病毒抑制最为强烈,且其CpG频率随年份呈下降趋势,但2009年毒株的CpG频率突然上升。比较同义密码子使用频率发现,含有CpG的同义密码子相对使用频率均小于1,证明CpG抑制作用是影响同义密码子偏爱性的一个重要因素。以上结果暗示,CpG抑制对H1N1流感病毒的进化及跨宿主传播可能有重要影响。     

The role of RNA folding free energy in the evolution of the polymerase genes of the influenza A virus

Background  

The influenza A virus genome is composed of eight single-stranded RNA segments of negative polarity. Although the hemagglutinin and neuraminidase genes are known to play a key role in host adaptation, the polymerase genes (which encode the polymerase segments PB2, PB1, PA) and the nucleoprotein gene are also important for the efficient propagation of the virus in the host and for its adaptation to new hosts. Current efforts to understand the host-specificity of the virus have largely focused on the amino acid differences between avian and human isolates.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.     

Rescue of influenza C virus from recombinant DNA

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.     

Nucleotide sequences of influenza virus segments 1 and 3 reveal mosaic structure of a small viral RNA segment

Defective interfering RNAs of influenza virus are small segments derived from viral segments 1, 2 and 3. We present here the complete nucleotide sequences of segments 1 and 3 from the human influenza strain A/PR/8/34 and deduce that the sequence of a small RNA segment from A/NT/60/68, apparently a defective interfering RNA, is derived from five separate regions in segment 3 and from one region in segment 1. These regions, which are located near the termini of the two parental segments, are arranged in the small RNA segment in an alternating fashion: thus a region derived from near a 5′ terminus is adjacent to a region derived from near a 3′ terminus. We propose that the small segment is generated during positive strand synthesis as a result of the viral polymerase pausing at uridine-rich sequences in the template and reinitiating synthesis at another site.     

Defective interfering virus associated with A/Chicken/Pennsylvania/83 influenza virus.

The A/Chicken/Pennsylvania/1/83 influenza virus, isolated from a respiratory infection of chickens, is an avirulent H5N2 virus containing subgenomic RNAs (W.J. Bean, Y. Kawaoka, J.M. Wood, J.E. Pearson, and R.G. Webster, J. Virol. 54:151-160, 1985). We show here that defective interfering particles are present in this virus population. The virus had a low ratio of plaque-forming to hemagglutinating units and produced interference with standard virus multiplication in infectious center reduction assays. Subgenomic RNAs were identified as internally deleted polymerase RNAs. We have confirmed that this virus protects chickens from lethal H5N2 influenza virus infection. This protective effect appeared to be due to the inhibition of virulent virus multiplication. Additionally, subgenomic RNAs derived from polymerase RNAs were detected in 5 of 18 RNA preparations from animal influenza virus isolates. Therefore, defective interfering particles are sometimes produced in natural influenza virus infections, not just under laboratory conditions. These particles may be capable of suppressing the pathogenic effect of virulent virus infections in nature.     

Influenza B virus genome: assignment of viral polypeptides to RNA segments.

It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural protein) were unambiguously assigned to specific RNA segments. Molecular weight estimates of the eight individual genes are obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.