Cell killing by viruses. II. Cell killing by vesicular stomatitis virus: a requirement for virion-derived transcription.
Cell killing by viruses. II. Cell killing by vesicular stomatitis virus: a requirement for virion-derived transcription.
复制标题
病毒杀死细胞。
DOI：
10.1016/0042-6822(75)90383-9
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发表时间：
1975
期刊：
影响因子：
3.7
通讯作者：
M. Sekellick
中科院分区：
文献类型：
--
作者：
P. I. Marcus;M. Sekellick
通过用印第安纳型水泡性口炎病毒（VSV）感染绿色猴肾（GMK）细胞的Vero细胞系生成的单细胞存活曲线，用于确定活性、UV辐照和热灭活（50°）病毒粒子以及在允许（30°）和非允许（40°）温度下测试的条件致死突变体的细胞杀伤颗粒（CKP）活性。将以这种方式处理的病毒体的CKP活性与体内测量的感染性、PFP和病毒体衍生的转录酶活性进行比较。热变性显示，所有这三种功能都以相同的速率丧失，绝对值显然是由病毒体相关转录酶的内在不稳定性决定的。对于VSV-HR和突变体ts G114，这些速率分别为K = 0.23 min− 1和K = 1.44 min−1。紫外线辐射灭活感染性（PFP）以指数速率与37%的存活剂量，D° = 52.3 ergs min−2。病毒体衍生的转录（体内测量）和CKP活性在照射后以一致的速率丧失，但对灭活的抵抗力提高了5倍-D ° = 263 ergs min−2。在所有情况下，体内病毒体衍生的转录酶和CKP活性以一致的速率被灭活。对于突变体ts G114（组I），在30°下CKP和PFP滴度相等。然而，ts G114在40°下不杀死细胞，也没有观察到显著的病毒体衍生的转录。相比之下，突变体ts W10（组IV）在30°-40°的温度范围内产生几乎相等的CKP滴度。尽管在放线菌酮存在下，大量初级转录物在30°下用ts G114感染的细胞中积累，但这些转录物在去除药物和温度转移到40°时不能有效地杀死细胞。突变体ts G114需要在30°下18-20小时以表达最大CKP活性。在30 ℃下6小时，50%的CKP的致死作用仍然存在，但只有不到10%的新合成病毒被释放。细胞杀伤因子在正常感染期间以及在非允许温度下细胞暴露于（1）缺陷型CKP、（2）紫外线照射的病毒体和（3）突变型ts W10时产生。病毒体衍生的初级转录是VSV产生细胞杀伤所必需的，但转录本本身的积累不足以杀死细胞。似乎推定的细胞杀伤因子的形成需要至少两种反应物：（1）初级转录物和（2）转录酶分子，其在初级转录之后的反应中起作用，其本质上可以是复制的或转录的。
Single-cell survival curves generated by infecting the Vero line of green monkey kidney (GMK) cells with Indiana-type vesicular stomatitis virus (VSV) were used to establish the cell killing particle (CKP) activity of active, uv-irradiated and heat-inactivated (50°) virions, as well as that of conditional-lethal mutants tested at permissive (30°) and nonpermissive (40°) temperatures. CKP activity of virions treated in this manner was compared to that of infectivity, PFP, and virion-derived transcriptase activity measuredin vivo. Heat denaturation revealed that all three of these functions were lost at the same rate, the absolute value apparently set by the intrinsic lability of the virion-associated transcriptase. For VSV-HR and mutant ts G114, these rates wereK= 0.23 min−1andK= 1.44 min−1, respectively. Ultraviolet-radiation inactivated infectivity (PFP) at an exponential rate with the 37% survival dose,D° = 52.3 ergs min−2. Virion-derived transcription as measuredin vivoand CKP activity were lost at coincident rates upon irradiation, but were 5 times more resistant to inactivation—D° = 263 ergs min−2. In all cases,in vivovirion-derived transcriptase and CKP activities were inactivated at coincident rates. With mutant ts G114 (group I), the CKP and PFP titers were equivalent at 30°. However, ts G114 did not kill cells at 40°, nor was significant virion-derived transcription observed. In contrast, mutant ts W10 (group IV) produced almost equivalent CKP titers over a temperature range of 30°–40°. Although large amounts of primary transcripts accumulate in the presence of cycloheximide in cells infected with ts G114 at 30°, these transcripts act inefficiently to kill cells upon removal of the drug and a temperature-shift to 40°. Mutant ts G114 requires 18–20 hr at 30° to express maximum CKP activity. The lethal action of 50% of the CKP is extant by 6 hr at 30°, yet less than 10% of newly synthesized virus has been released. Cell killing factor is produced during normal infection and upon exposure of cells to (1) defective CKP, (2) uv-irradiated virions and (3) mutant ts W10 at nonpermissive temperatures. Virion-derived primary transcription is required to produce cell killing by VSV, but accumulation of transcripts per se does not suffice to kill cells. It appears likely that formation of a putative cell killing factor requires at least two reactants: (1) primary transcripts and (2) transcriptase molecules, functioning in a reaction(s) subsequent to primary transcription which could be replicative or transcriptive in nature.