Influenza viral messenger RNA

Influenza viral messenger RNA (mRNA) free from both ribosomal RNA and newly synthesized host mRNA was isolated from the polyribosomes of infected canine kidney cells. Cordycepin was added to infected cells to inhibit ribosomal RNA and host mRNA synthesis. To separate viral mRNA from the small amount of ribosomal RNA which continued to be synthesized in the presence of cordycepin, polyribosomes were dissociated with puromycin and high salt: the viral mRNA was released to sediment 8–22 S, thus separable from the ribosomal RNA which was quantitatively retained in the 60 S and 40 S ribosomal subunits.Viral mRNA was rendered 100% ribonuclease-resistant after annealing to virion RNA (vRNA) and is, therefore, totally complementary to vRNA. This complementary RNA (cRNA) contains polyadenylate (polyA) segments which are heterogeneous in length, ranging from 50 to 200 nucleotides. In contrast to cRNA, vRNA does not contain polyA.     

Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates

To identify the potential RNA inhibitors of HCV polymerase, we have isolated high-affinity RNA ligands specific to hepatitis C virus (HCV) NS5B protein from a combinatorial RNA library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) procedure. Thirty-seven selected ligands were classified into eight groups on the basis of their sequence homologies. Most (60%) of the ligands carry the conserved YGUAGR hexamer (Y = pyrimidine, R = purine) at the 5' end of the 40-nt randomized region, and 74% of the ligands end in (A/C)U at the 3'end. However, strong binding to NS5B required the whole RNA ligand including the flanking conserved nucleotides at both ends. The binding of the selected ligands to NS5B is highly specific and strong, as reflected in their low dissociation rate constants (k(d) approximately 10(-4) s(-1)). Analysis of secondary structure by computer program and RNase footprints of the two different aptamers from two most conserved groups revealed RNA structures containing three stem loops with internal bulges. NS5B bound these RNA at a region between the two stem loops from the 5' -end. Some of these RNA aptamers could serve as a template for the HCV polymerase, but some interfered with the activity of the viral enzyme. These RNA ligands will be useful for further characterization of NS5B-binding properties and, with further modifications, may have potential therapeutic value.     

Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.     

Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

In vitro, the coat protein of brome mosaic virus (BMV) inhibited RNA synthesis by replicase extracted from BMV-infected barley leaves. The inhibition was due to the interaction of coat protein with BMV RNA. Under the same conditions, no inhibition by TMV coat protein or bovine serum albumin was detected. For the complete inhibition of RNA synthesis in vitro, a coat protein:RNA ratio of 620:1 was required, and their preincubation before addition to the reaction mixture was essential. If the coat protein was added to the reaction mixture during incubation, synthesis continued for a few minutes at the level of the control (omission of coat protein), then decreased gradually, and stopped 6 min after the addition of coat protein. These results suggest that the inhibitory effect of coat protein on RNA synthesis in vitro is due to the interference with the binding site of replicase by partial reassembly of nucleoprotein and that this phenomenon may be a cause of cross protection.     

Hepatitis C virus evasion of adaptive immune responses: a model for viral persistence

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. Approximately 80% of those acutely infected clear the infection, whereas the remaining 20% progress to chronic infection. Hepatitis C thus provides a model in which successful and unsuccessful responses can be compared to better understand the human response to viral infection. Our laboratory studies the strategies by which HCV evades the adaptive immune response. This review describes the impact of viral mutation on T cell recognition, the role of cell surface inhibitory receptors in recognition of HCV, and the development of antibodies that neutralize HCV infection. Understanding what constitutes an effective immune response in the control of HCV may enable the development of prophylactic and therapeutic vaccines for HCV and other chronic viral infections.     

Replicative forms and viral RNA synthesis in leaves infected with brome mosaic virus

Four major RNAs (A, A′, B, D) and one minor RNA (C) have been isolated by polyacrylamide gel electrophoresis from purified brome mosaic virus. All RNA components were stable when treated with heat, DMSO, and formamide, thus excluding the possibility of hidden breaks. The mixture A + A′ + B was infectious. Analysis of the replicative forms (RF) isolated from infected plants showed that only the three largest RNAs (A, A′, B) had their own RFs. ³²P incorporation showed that the viral RNAs were synthesized at different rates. The specific activities of A + A′ and D were identical and were always higher than that of B. The minor component C was rapidly labeled but very little was encapsulated. The origin and significance of RNAs C and D are discussed.     

核酶在细胞外切割HCV核心区RNA的研究

目的 研究核酶在细胞外切割HCV的作用。方法 设计核酶cDNA序列,构建核酶重组质粒及HCV核心区基因cDNA重组质粒。分别进行体外转录,并加入γ-^32P ATP以标记HCV RNA。将核酶RNA、HCV核心区RNA、RNA酶抑制剂及反应缓冲液混事浊国育,以核酶RNA切割HCV RNA。通过变性聚 烯酰胺凝胶电泳及放射自显影来分析结果。结果 β－半乳糖苷酶表型筛选均可见蓝色及白色菌落生长;核酶重组质粒直接测序结果见预期要苷酸序列;HCV重组质粒限制笥酶切分析见410bp条带。核酶切割反应显示：反应时间为15和30min时可见453、307、146nt3条带。反应时间为60min时仅见307及146nt2条带。结论 核酶重组质粒构建成功,所设计核酶对HCV有切割作用。     

Evasion of host immune surveillance by hepatitis C virus: potential roles in viral persistence

Hepatitis C virus (HCV) is a major human pathogen that causes mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. In order for a noncytopathic virus such as HCV to persist, the virus must escape immune recognition or evade host immune surveillance. Immune escape via the hypervariable region of the E2 envelope protein has been postulated as one mechanism for HCV persistent infection. Such hypervariability within the E2 protein may be under selective pressure from protective B cell or T cell responses and be able to escape immune recognition by rapid mutation of antigenic site. In addition to antigenic variation, HCV may also suppress immune response, leading to dampening of cellular immunity. This is supported by recent studies in our laboratory demonstrating that the HCV core protein can suppress host immune responses to vaccinia virus by downregulating viral specific cytotoxic T lymphocyte (CTL) responses and cytokine production. An understanding of the mechanisms behind HCV persistence will provide a basis for the rational design of vaccines and novel therapeutic agents targeting human HCV infection.