Schang L M, Rosenberg A, Schaffer P A
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
J Virol. 2000 Mar;74(5):2107-20. doi: 10.1128/jvi.74.5.2107-2120.2000.
We have previously shown that two inhibitors specific for cellular cyclin-dependent kinases (cdks), Roscovitine (Rosco) and Olomoucine (Olo), block the replication of herpes simplex virus (HSV). Based on these results, we demonstrated that HSV replication requires cellular cdks that are sensitive to these drugs (L. M. Schang, J. Phillips, and P. A. Schaffer. J. Virol. 72:5626-5637, 1998). We further established that at least two distinct steps in the viral replication cycle require cdks: transcription of immediate-early (IE) genes and transcription of early (E) genes (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Since Rosco inhibits HSV replication efficiently even when added to infected cells at 6 h postinfection, we postulated that cdks may also be required for viral functions that occur after E gene expression. In the study presented herein, we tested this hypothesis directly by measuring the efficiency of viral replication, viral DNA synthesis, and expression of several viral genes during infections in which Rosco was added after E proteins had already been synthesized. Rosco inhibited HSV replication, and specifically viral DNA synthesis, when the drug was added at the time of release from a 12-h phosphonoacetic acid (PAA)-induced block in viral DNA synthesis. Inhibition of DNA synthesis was not a consequence of inhibition of expression of IE or E genes in that Rosco had no effect on steady-state levels of two E transcripts under the same conditions in which it inhibited viral DNA synthesis. Moreover, viral DNA synthesis was inhibited by Rosco even in the absence of protein synthesis. In a second series of experiments, the replication of four HSV mutants harboring temperature-sensitive mutations in genes essential for viral DNA replication was inhibited when Rosco was added at the time of shift-down from the nonpermissive to the permissive temperature. Viral DNA synthesis was inhibited by Rosco under these conditions, whereas expression of viral E genes was not affected. We conclude that cellular Rosco-sensitive cdks are required for replication of viral DNA in the presence of viral E proteins. This requirement may indicate that HSV DNA synthesis is functionally linked to transcription, which requires cdks, or that both viral transcription and DNA replication, independently, require viral or cellular factors activated by Rosco-sensitive cdks.
我们之前已经表明，两种细胞周期蛋白依赖性激酶（cdks）特异性抑制剂，即Roscovitine（Rosco）和Olomoucine（Olo），可阻断单纯疱疹病毒（HSV）的复制。基于这些结果，我们证明HSV复制需要对这些药物敏感的细胞cdks（L. M. Schang、J. Phillips和P. A. Schaffer。《病毒学杂志》72:5626 - 5637，1998年）。我们进一步确定，病毒复制周期中至少有两个不同步骤需要cdks：立即早期（IE）基因的转录和早期（E）基因的转录（L. M. Schang、A. Rosenberg和P. A. Schaffer，《病毒学杂志》73:2161 - 2172，1999年）。由于即使在感染后6小时添加到受感染细胞中，Rosco也能有效抑制HSV复制，我们推测在E基因表达后发生的病毒功能可能也需要cdks。在本文所述的研究中，我们通过在E蛋白已经合成后添加Rosco的感染过程中，测量病毒复制效率、病毒DNA合成以及几种病毒基因的表达，直接检验了这一假设。当在从12小时的膦甲酸（PAA）诱导的病毒DNA合成阻断中释放时添加该药物，Rosco抑制了HSV复制，特别是病毒DNA合成。DNA合成的抑制不是IE或E基因表达受到抑制的结果，因为在抑制病毒DNA合成的相同条件下，Rosco对两种E转录本的稳态水平没有影响。此外，即使在没有蛋白质合成的情况下，Rosco也能抑制病毒DNA合成。在第二系列实验中，当在从非允许温度转变到允许温度时添加Rosco，四个在病毒DNA复制所必需的基因中携带温度敏感突变的HSV突变体的复制受到抑制。在这些条件下，Rosco抑制了病毒DNA合成，而病毒E基因的表达未受影响。我们得出结论，在存在病毒E蛋白的情况下，细胞中对Rosco敏感的cdks是病毒DNA复制所必需的。这一需求可能表明HSV DNA合成在功能上与转录相关联，转录需要cdks，或者病毒转录和DNA复制独立地需要由对Rosco敏感的cdks激活的病毒或细胞因子。
