RESUMO
Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.
Assuntos
Dependovirus/química , Vetores Genéticos/normas , Guias de Prática Clínica como Assunto , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Criopreservação/normas , Dependovirus/genética , Dependovirus/fisiologia , Vetores Genéticos/química , Células HEK293 , Humanos , Estabilidade Proteica , Padrões de Referência , Replicação ViralRESUMO
The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication.
Assuntos
Poliovirus/fisiologia , Precursores de Proteínas/metabolismo , RNA Viral/genética , Proteínas Virais/metabolismo , Replicação Viral/genética , Ordem dos Genes , Teste de Complementação Genética , Modelos Biológicos , Mutação , Ligação Proteica , Precursores de Proteínas/genética , RNA Viral/química , Proteínas Virais/genéticaRESUMO
The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol).
Assuntos
Amilorida/farmacologia , Antivirais/farmacologia , Enterovirus/efeitos dos fármacos , Enterovirus/genética , Poliovirus/efeitos dos fármacos , Poliovirus/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Amilorida/química , Antivirais/química , Infecções por Coxsackievirus/virologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Enterovirus/química , Enterovirus/fisiologia , Humanos , Modelos Moleculares , Poliomielite/virologia , Poliovirus/química , Poliovirus/fisiologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The genomic RNA of poliovirus and closely related picornaviruses perform template and non-template functions during viral RNA replication. The non-template functions are mediated by cis-active RNA sequences that bind viral and cellular proteins to form RNP complexes. The RNP complexes mediate temporally dynamic, long-range interactions in the viral genome and ensure the specificity of replication. The 5' cloverleaf (5' CL)-RNP complex serves as a key cis-active element in all of the non-template functions of viral RNA. The 5'CL-RNP complex is proposed to interact with the cre-RNP complex during VPgpUpU synthesis, the 3'NTR-poly(A) RNP complex during negative-strand initiation and the 30 end negative-strand-RNP complex during positive-strand initiation. Co-ordinating these long-range interactions is important in regulating each step in the replication cycle.
Assuntos
Picornaviridae/fisiologia , RNA Viral/metabolismo , Replicação Viral , Replicação do DNA , Picornaviridae/enzimologia , Picornaviridae/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Moldes Genéticos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In this study, we showed that the 5'CL-PCBP complex, 3' poly(A) tail and viral protein 2A(pro) are all required for optimal translation of PV RNA. The 2A(pro)-mediated stimulation of translation was observed in the presence or absence of both the 5'CL and the 3' poly(A) tail. Using protein-RNA tethering, we established that the 5'CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5'CL-PCBP complex and the 3' poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5'CL and the 3' poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5'CL-PCBP complex interacts with the 3' poly(A)-PABP complex to form a 5'-3' circular complex that facilitates ribosome reloading and stimulates PV RNA translation.
Assuntos
Cisteína Endopeptidases/metabolismo , Poliovirus/fisiologia , Biossíntese de Proteínas , RNA Mensageiro , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Ligação ProteicaRESUMO
Using cell-free reactions, we investigated the role of the 5' cloverleaf (5'CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop 'b' and the C-rich sequence was inhibited. Taken together, these findings suggest that PCBP binding to the C-rich sequence was sufficient to support RNA stability and negative-strand synthesis. Mutational analysis of the three conserved structural elements in stem-loop 'd' showed that they were required for efficient negative- and positive-strand synthesis. Finally, we showed an RNA with a 5' terminal deletion (Delta49TD RNA), which was previously isolated from persistently infected cells, replicated at low but detectable levels in these reactions. Importantly, the critical replication elements identified in this study are still present in the Delta49TD RNA.
Assuntos
Enterovirus/fisiologia , Estabilidade de RNA , RNA Viral/biossíntese , Replicação Viral , Proteínas de Ligação a DNA , Enterovirus/genética , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/genética , Proteínas de Ligação a RNARESUMO
Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Regulação Viral da Expressão Gênica , Poliovirus/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Cisteína Endopeptidases/genética , Células HeLa , Humanos , Poliovirus/genética , RNA Viral/química , RNA Viral/genética , Proteínas Virais/genética , Replicação ViralRESUMO
The precise relationship between the length of the 3' poly(A) tail and the replication and infectivity of poliovirus RNA was examined in this study. With both poly(A)(11) and poly(A)(12) RNAs, negative-strand synthesis was 1-3% of the level observed with poly(A)(80) RNA. In contrast, increasing the length of the poly(A) tail from (A)(12) to (A)(13) resulted in about a ten-fold increase in negative-strand synthesis. This increase continued with each successive increase in poly(A) tail length. With poly(A)(20) RNA, RNA synthesis approached the level observed with poly(A)(80) RNA. A similar relationship was observed between poly(A) tail length and the infectivity of the viral RNA. A replication model is described which suggests that viral RNA replication is dependent on a poly(A) tail that is long enough to bind poly(A) binding protein and to act as a template for VPg uridylylation and negative-strand initiation.