Ansardi D C, Pal-Ghosh R, Porter D, Morrow C D
Department of Microbiology, University of Alabama at Birmingham 35294.
J Virol. 1995 Feb;69(2):1359-66. doi: 10.1128/JVI.69.2.1359-1366.1995.
The multiple roles of the viral proteinase 2A in poliovirus replication have been difficult to assess because, to date, it has not been possible to isolate and characterize a viral genome with an inactive 2Apro. We have previously reported that a poliovirus replicon containing an inactive 2Apro by virtue of a change at amino acid 109 from a cysteine to a serine (C109S) was replication competent when transfected into cells previously infected with vaccinia virus (R. Pal-Ghosh and C. D. Morrow, J. Virol. 67:4621-4629, 1993). To further develop this system, we have used a poliovirus replicon which contains the human immunodeficiency virus type 1 (HIV-1) gag gene positioned between nucleotides 1174 and 2470 of the poliovirus genome and have engineered a second mutation within this replicon to change the codon for amino acid 109 of the 2Apro from cysteine to serine (2AC109S). Transfection of this replicon into cells previously infected with vaccinia virus results in the replication and expression of a protein with a molecular mass consistent with that of a P1-HIV-1 Gag-2A fusion protein. Using a recently described complementation system which relies on the capacity of a recombinant vaccinia virus (VV-P1) to provide the capsid precursor (P1) in trans (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993; and D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993), we have encapsidated this replicon containing the 2AC109S mutation. By using reverse transcription PCR, we demonstrated that after 15 serial passages the encapsidated replicon still contained the 2AC109S mutation. Infection of cells with a stock of encapsidated replicon, either in the presence or in the absence of vaccinia virus, resulted in the expression of the P1-HIV-1 Gag-2A fusion protein. Expression of the P1-HIV-1 Gag fusion protein in cells infected with the encapsidated replicon containing the 2AC109S mutation was reduced compared with the expression of P1-HIV-1 Gag in those cells infected with a replicon containing a wild type 2A gene. The protein expression and replication of the replicon RNA in cells containing the 2AC109S mutation was maintained for a longer period of time than for the replicons containing the wild-type 2A gene, possibly because of a reduced cytopathic effect.(ABSTRACT TRUNCATED AT 400 WORDS)
脊髓灰质炎病毒蛋白酶2A在脊髓灰质炎病毒复制中的多种作用一直难以评估，因为迄今为止，还无法分离和鉴定具有无活性2A蛋白酶的病毒基因组。我们之前报道过，一种脊髓灰质炎病毒复制子，由于其109位氨基酸由半胱氨酸突变为丝氨酸（C109S）而导致2A蛋白酶无活性，当转染到先前感染痘苗病毒的细胞中时，它具有复制能力（R. 帕尔 - 戈什和C. D. 莫罗，《病毒学杂志》67:4621 - 4629，1993年）。为了进一步开发这个系统，我们使用了一种脊髓灰质炎病毒复制子，它在脊髓灰质炎病毒基因组的1174和2470核苷酸之间包含人类免疫缺陷病毒1型（HIV - 1）gag基因，并在这个复制子内设计了第二个突变，将2A蛋白酶109位氨基酸的密码子由半胱氨酸变为丝氨酸（2AC109S）。将这个复制子转染到先前感染痘苗病毒的细胞中，会导致一种分子量与P1 - HIV - 1 Gag - 2A融合蛋白一致的蛋白质的复制和表达。使用最近描述的一种互补系统，该系统依赖于重组痘苗病毒（VV - P1）反式提供衣壳前体（P1）的能力（D. C. 安萨尔迪、D. C. 波特和C. D. 莫罗，《病毒学杂志》67:3684 - 3690，1993年；以及D. C. 波特、D. C. 安萨尔迪、W. S. 崔和C. D. 莫罗，《病毒学杂志》67:3712 - 3719，1993年），我们将这个含有2AC109S突变的复制子进行了衣壳化。通过逆转录PCR，我们证明在连续传代15次后，衣壳化的复制子仍然含有2AC109S突变。用衣壳化复制子的储备液感染细胞，无论有无痘苗病毒，都会导致P1 - HIV - 1 Gag - 2A融合蛋白的表达。与用含有野生型2A基因的复制子感染的细胞相比，用含有2AC109S突变的衣壳化复制子感染的细胞中P1 - HIV - 1 Gag融合蛋白的表达减少。含有2AC109S突变的复制子RNA在细胞中的蛋白质表达和复制比含有野生型2A基因的复制子维持的时间更长，这可能是由于细胞病变效应降低所致。（摘要截短至400字）
