Mavromara-Nazos P, Roizman B
Marjorie B. Kovler Viral Oncology Laboratory, University of Chicago, IL 60637.
Proc Natl Acad Sci U S A. 1989 Jun;86(11):4071-5. doi: 10.1073/pnas.86.11.4071.
The expression of the gamma 2 class of viral genes in cells infected with herpes simplex virus 1 requires viral DNA synthesis and functional viral products made earlier in infection. To identify the sequences required for gamma 2 gene expression, we constructed recombinant viruses in which regions of the thymidine kinase gene (tk), a beta gene normally expressed early in infection, were replaced by specific domains of a gamma 2 gene. The phenotypic attributes examined were (i) sensitivity or resistance of expression in cells exposed to sufficient phosphonoacetate to block viral DNA synthesis, properties of gamma 2 and beta genes, respectively; (ii) expression early in infection, a property of beta genes; and (iii) expression late in infection, a property of gamma 2 genes. We report that replacement of nucleotides -200 to +51 of tk with nucleotides -77 to +104 of the gamma 2 gene conferred upon tk all of the tested attributes of gamma 2 genes. The tk sequence in the 5' transcribed noncoding domain downstream of nucleotide +51 played no apparent role in the expression of the chimeric genes. Similarly, tk sequence downstream of -16 and gamma 2 sequence upstream of -12, when juxtaposed in correct orientations, yielded a chimeric gene that was poorly expressed. In contrast, the chimeric gene consisting of tk sequence upstream of -16 fused to gamma 2 sequence downstream of -12 had the attributes of both beta and gamma 2 genes in that it was expressed both early and late in infection and was partially resistant to phosphonoacetate. The capacity for expression late in infection encoded in the gamma 2 5' transcribed noncoding domain was observed in cells infected with a recombinant virus in which gamma 2 nucleotides +17 to +104 were inserted into the 5' transcribed noncoding domain of the tk gene. We conclude that whereas in the beta genes exemplified by the tk gene the regulatory domains are mainly upstream from nucleotide +51, the sequence(s) that confers gamma 2 regulation is downstream from the TATAA box.
在感染单纯疱疹病毒1的细胞中，病毒γ2类基因的表达需要病毒DNA合成以及感染早期产生的功能性病毒产物。为了鉴定γ2基因表达所需的序列，我们构建了重组病毒，其中胸苷激酶基因（tk）（一种通常在感染早期表达的β基因）的区域被γ2基因的特定结构域所取代。所检测的表型特征包括：（i）在暴露于足以阻断病毒DNA合成的膦乙酸的细胞中表达的敏感性或抗性，分别为γ2和β基因的特性；（ii）感染早期的表达，β基因的特性；以及（iii）感染后期的表达，γ2基因的特性。我们报告，用γ2基因的核苷酸-77至+104替换tk的核苷酸-200至+51赋予了tk所有测试的γ2基因属性。核苷酸+51下游5'转录非编码结构域中的tk序列在嵌合基因的表达中未发挥明显作用。同样，-16下游的tk序列和-12上游的γ2序列以正确方向并列时，产生的嵌合基因表达不佳。相反，由-16上游的tk序列与-12下游的γ2序列融合而成的嵌合基因具有β和γ2基因的属性，因为它在感染早期和后期均有表达，并且对膦乙酸有部分抗性。在感染一种重组病毒的细胞中观察到γ2 5'转录非编码结构域中编码的感染后期表达能力，在该重组病毒中，γ2核苷酸+17至+104插入到tk基因的5'转录非编码结构域中。我们得出结论，以tk基因为例的β基因中，调控结构域主要在核苷酸+51上游，而赋予γ2调控的序列在TATAA框下游。
