Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus.

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

Duplications of a mutated simian virus 40 enhancer restore its activity

Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.     

Recognition and initiation site for four late promoters of phage T7 is a 22-base pair DNA sequence

T7 late promoters have a 22-base pair sequence in common. The 22 base pairs are necessary and sufficient for recognition and initiation by T7 RNA polymerase.     

p53 and DNA polymerase alpha compete for binding to SV40 T antigen

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.     

2'5' oligo(A) polymerase activity in serum of mice infected with EMC virus or treated with interferon

Interferon-treated cells show an increase in two double-stranded RNA (dsRNA)-dependent enzymatic activities involving an oligoadenylate polymerase and a protein kinase (ref. 1 and refs therein). The polymerase converts ATP into a series of oligonucleotides characterized by 2'5'-phosphodiester bonds, designated 2'5'-oligo(A) or 2-5A (ref. 1). These oligonucleotides activate an endoribonuclease that degrades RNA in extracts of control and interferon-treated cells. These observations have been made in tissue culture cells and no informatin is yet available on these enzymatic activities in animals with elevated interferon levels. We report here on 2-5A synthesis in tissue homogenates and serum of mice infected with encephalomyocarditis virus (EMCV); this virus induces interferon synthesis when injected intraperitoneally into mice. Significant synthesis of 2-5A was detected in extracts of spleen and lungs, but also, surprisingly, in the serum of these mice. Subsequent experiments showed synthesis of 2-5A in serum of mice treated with the interferon inducer poly(I) x poly(C) (ref. 3) or with mouse fibroblast interferon.     

Molecular virology: was the 1918 pandemic caused by a bird flu?

Taubenberger et al. have sequenced the polymerase genes of the pandemic 'Spanish' influenza A virus of 1918, thereby completing the decoding of the genome of this virus. The authors conclude from these sequences that the virus jumped from birds to humans shortly before the start of the pandemic and that it was not derived from earlier viruses by gene shuffling, a process called reassortment. However, we believe that their evidence does not convincingly support these conclusions and that some of their results even indicate that, on the contrary, the virus evolved in mammals before the pandemic began and that it was a reassortant. In light of this alternative interpretation, we suggest that the current intense surveillance of influenza viruses should be broadened to include mammalian sources.     

金银花U6启动子的克隆及转录活性分析

U6启动子是构建CRISPR/Cas9体系中驱动sgRNA转录的重要元件。利用聚合酶链式反应方法,从华金6号金银花中克隆到3种U6启动子,并分别构建5个不同长度的启动子驱动GUS的植物融合表达载体。以CaMV35S作为阳性对照实验,农杆菌作为阴性对照实验,采用农杆菌瞬时转化法对烟草原生质体进行转染,GUS染色后置于显微镜下观察原生质体的染色数量和效率。根据染色结果,成功筛选出Chr01 U6-F1这一高效转录的启动子,为后期开展金银花的基因编辑奠定了基础。