When human immunodeficiency virus type 1 (HIV-1) is selected for resistance to 3TC, the methionine normally present at position 184 is replaced by valine or isoleucine. Position 184 is the X of the conserved YXDD motif; positions 185 and 186 form part of the triad of aspartic acids at the polymerase active site. Structural and biochemical analysis of 3TC-resistant HIV-1 reverse transcriptase (RT) led to a model in which a -branched amino acid at position 184 would act as a steric gate. Normal deoxynucleoside triphosphates (dNTPs) could still be incorporated; the oxathiolane ring of 3TCTP would clash with the  branch of the amino acid at position 184. This model can also explain 3TC resistance in feline immunodeficiency virus and human hepatitis B virus. However, it has been reported (14) that murine leukemia viruses (MLVs) with valine (the amino acid present in the wild type), isoleucine, alanine, serine, or methionine at the X position of the YXDD motif are all resistant to 3TC. We prepared purified wild-type MLV RT and mutant MLV RTs with methionine, isoleucine, and alanine at the X position. The behavior of these RTs was compared to those of wild-type HIV-1 RT and of HIV-1 RT with alanine at the X position. If alanine is present at the X position, both MLV RT and HIV-1 RT are relatively resistant to 3TCTP in vitro. However, the mutant enzymes were impaired relative to their wild-type counterparts; there appears to be steric hindrance for both 3TCTP and normal dNTPs.Considerable progress has been made in the development of anti-human immunodeficiency virus type 1 (HIV-1) drugs and drug therapies. However, the emergence of drug-resistant viral strains presents a major problem; understanding the mechanisms that underlie drug resistance should be an important part of the effort to develop more effective drugs. Most of the available drugs target one of two viral enzymes, reverse transcriptase (RT) and protease (PR). There are two classes of RT inhibitors, nucleoside analogs and nonnucleosides. The nucleoside analogs used to treat HIV-1 infections lack the normal 3Ј OH of the ribose ring. The compounds are usually given to patients in an unphosphorylated state. The compounds are taken up by cells and converted to triphosphates by cellular enzymes. In this form, the analogs can be incorporated into viral DNA by HIV-1 RT; once incorporated, nucleoside analogs act as chain terminators, blocking viral DNA synthesis. One of the nucleoside analogs commonly used to treat HIV-1 infections is 3TC. 3TC treatment selects for drug-resistant viruses that have the methionine normally present at position 184 replaced either by isoleucine or valine. Viruses that have either the M184I or M184V mutation are quite resistant to 3TC; purified recombinant HIV-1 RTs that carry these mutations are resistant to 3TCTP in simple in vitro polymerization assays (9, 11,18).Nucleoside analogs inhibit reverse transcription (and viral replication) because they are incorporated into viral DNA by HIV-1 RT. Resistance to nucleoside analogs implies ...
The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.
We have analyzed amino acid substitutions at position G190 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The mutation G190E, which is responsible for resistance to certain nonnucleoside inhibitors, results in RT that has significantly less polymerase activity and that is less processive than wild-type RT. Its kinetic profile with respect to dGTP and poly(rC) · oligo(dG) is significantly altered compared to that of wild-type RT. The combination of either of the mutations L74V or V75I with the G190E mutation appears to be compensatory and mitigates many of the deleterious effects of the G190E mutation.
Escherichia coli RNase H has a basic extension that is involved in binding nucleic acid substrates. This basic extension is present in the RNase H of Moloney murine leukemia virus reverse transcriptase (MLV RT), but has been deleted from the RNase H of HIV-1 RT. Previous work showed that removing the basic loop from MLV RT (the mutant is called DeltaC) blocked viral replication; however, DeltaC MLV RT retained RNase H activity in an in situ gel assay. We prepared recombinant DeltaC MLV RT and compared its activity to wild-type MLV RT. The DeltaC mutant is impaired in both polymerase and RNase H activity; the pattern of defects suggests that the basic loop is involved in the binding of MLV RT to a heteropolymeric template-primer.
Objective: has been shown to participate in multiple malignancies, but the role of miR-203 in hepatoblastoma (HB) remains unclear. The aim of our study was to investigate the effects of miR-203 in HB. Methods: A total of 15 pairs of HB tissues and para-tumour normal tissues were collected for the experiments. RT-qPCR and Western blotting were performed to detect the expression of CRNDE, miR-203, and VEGFA at the mRNA and/or protein levels, respectively. A dual luciferase assay verified the target relationship between miR-203 and the 3′UTR of VEGFA as well as miR-203 and CRNDE. In addition, MTT, wound healing, and tube formation assays were performed to assess the effects of miR-203, VEGFA, and CRNDE on cell proliferation, migration, and angiogenesis, respectively. Results: Our data revealed that miR-203 expression was decreased in HB tissues, while long non-coding RNA (lncRNA) CRNDE expression was increased. The dysregulation of miR-203 and CRNDE was closely related to tumour size and stage. Moreover, overexpression of miR-203 inhibited angiogenesis. A dual luciferase assay verified that VEGFA is a direct target of miR-203 and that CRNDE binds to miR-203. Furthermore, our results showed that miR-203 suppressed cell viability, migration, and angiogenesis by regulating VEGFA expression. Additionally, it was confirmed that CRNDE promoted angiogenesis by negatively regulating miR-203 expression. Conclusion: lncRNA CRNDE targets the miR-203/VEGFA axis and promotes angiogenesis in HB. These results provide insight into the underlying mechanisms of HB and indicate that CRNDE and miR-203 might be potential targets for HB therapy.
The synthesis of retroviral DNA is initiated near the 5 end of the RNA. DNA synthesis is transferred from the 5 end to the 3 end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5 cap, to investigate the rules governing RNase H cleavage at the 5 end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.The retroviral genome is single-stranded RNA. When the virus infects a susceptible cell, the core enters the cytoplasm and genomic RNA is converted into linear double-stranded DNA by the viral enzyme reverse transcriptase (RT) (reviewed in references 3, 11, and 19). RT has two enzymatic activities: a DNA polymerase that can copy either an RNA or a DNA template, and RNase H, which will cleave RNA if, and only if, it is part of an RNA/DNA duplex. These two activities collaborate in the conversion of the RNA genome into DNA. RT, like many other DNA polymerases, requires both a template and a primer. Reverse transcription is initiated from a host tRNA base-paired to the viral genome at the primer binding site near the 5Ј end of the viral genome. First-strand DNA synthesis creates an RNA/DNA duplex; this duplex is a substrate for RNase H.Because the primer binding site is near the 5Ј end of the RNA genome, DNA synthesis rapidly reaches the end of the RNA. This DNA is called minus-strand strong-stop DNA (ϪsssDNA). DNA synthesis is then transferred to the 3Ј end of the RNA genome. This transfer reaction requires that RNase H degrade the 5Ј end of the RNA genome. The 5Ј ends of the genomes of several complex retroviruses (human immunodeficiency virus type 1 [HIV-1], HIV-2, and human T-cell leukemia virus type 2) can form complex secondary structures; in the absence of the complementary RNA, the newly synthesized ϪsssDNAs of these retroviruses can fold into complex structures. Some of these complex DNA structures have the potential to self-prime. Self-priming is prevented in vivo; the process appears to involve both the nucleocapsid (NC) protein and a piece of viral RNA from the 5Ј end of the genome (5, 7, 10). The available data, most of which were obtained with in vitro reactions using model substrates, suggest that a residual piece from the 5Ј end of the RNA genome prevents selfpriming. To block self-priming, the residual RNA must be long enough so that when it is annealed to ϪsssDNA, the RNA is able to pr...
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