Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.

核出口是疱疹病毒复制的一个限速步骤，它限制了病毒衣壳的核质运输。该过程受两种病毒核外出蛋白（核心NEC pUL50-pUL53）的调节，它们吸收了其他细胞和病毒蛋白。多组分NEC介导核层屏障的拆卸和核衣壳的对接。核出口的定量已经通过电子显微镜分析完成，但是通常由于可检测的胞质衣壳数量少而受到阻碍。一种新建立的量化病毒核出口的方法改善了病毒突变体的表征，宿主细胞的允许性和抗病毒药物的功效。在这个研究中，使用各种人类巨细胞病毒株（HCMV）来测量人类原代成纤维细胞的复制效率，细胞分级分离，DNA酶消化，蔗糖缓冲液和定量PCR的方法。优化的几个阶段导致了可靠的定量分析，该分析可以表征病毒核出口功效。使用该测定法，通过应用可表达的表达NEC的成纤维细胞培养物进行反式互补，定量评估NEC缺陷HCMV突变体核出口的恢复。这种新颖的测定系统可进一步用于准确定量和表征HCMV或其他疱疹病毒的核出口功能。优化的几个阶段导致了可靠的定量分析，该分析可以表征病毒核出口功效。使用该测定法，通过应用可表达的表达NEC的成纤维细胞培养物进行反式互补，定量评估NEC缺陷HCMV突变体核出口的恢复。这种新颖的测定系统可进一步用于准确定量和表征HCMV或其他疱疹病毒的核出口功能。优化的几个阶段导致了可靠的定量分析，该分析可以表征病毒核出口功效。使用该测定法，通过应用可表达的表达NEC的成纤维细胞培养物进行反式互补，定量评估NEC缺陷HCMV突变体核出口的恢复。这种新颖的测定系统可进一步用于准确定量和表征HCMV或其他疱疹病毒的核出口功能。