Li Maggie, Smith Barbara, Jaeyeun Lee, Petr Jennifer, Wiseman Robyn, Anders Nicole, Rudek Michelle, Ambinder Richard, Desai Prashant
Johns Hopkins University.
Res Sq. 2023 Nov 8:rs.3.rs-3552962. doi: 10.21203/rs.3.rs-3552962/v1.
Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) infections aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions.
For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication.
We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 minutes. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein ORF26 was markedly reduced. This is an essential protein required for herpesvirus capsid assembly.
Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.
靶向疱疹病毒的抗病毒疗法在临床上具有重要意义。奈非那韦是一种蛋白酶抑制剂，可靶向人类免疫缺陷病毒（HIV）感染的天冬氨酸蛋白酶。先前的研究表明，这种药物还可以抑制卡波西肉瘤相关疱疹病毒（KSHV）的产生。我们实验室证明奈非那韦可以有效抑制单纯疱疹病毒1型（HSV-1）的复制。对于HSV-1，我们能够确定病毒衣壳已组装并离开细胞核，但在细胞质中未成熟，这表明该药物抑制了病毒粒子的二次包膜化。
对于KSHV，我们最近建立了一个易于处理的细胞培养系统，使我们能够详细分析病毒复制周期。我们使用这个系统来进一步确定奈非那韦抑制KSHV复制的阶段。
我们发现奈非那韦抑制KSHV细胞外病毒的产生。当药物与细胞孵育3天时以及当我们用药物脉冲细胞1 - 5分钟时，都观察到了这种情况。当使用超微结构方法检查暴露于该药物的KSHV感染细胞时，细胞核中没有成熟的衣壳，这表明衣壳组装存在缺陷。由于奈非那韦会影响整合应激反应（ISR），我们在药物存在的情况下检查了病毒蛋白的表达。我们观察到许多病毒蛋白的表达在药物存在时发生了显著变化。衣壳三聚体蛋白ORF26的积累明显减少。这是疱疹病毒衣壳组装所需的一种必需蛋白。
我们的研究证实奈非那韦通过破坏病毒组装和成熟来抑制KSHV病毒粒子的产生。有趣的是，抑制作用仅需要短时间接触药物。唾液中传染性病毒的来源尚未详细确定，但很可能是口腔黏膜中的淋巴细胞或其他细胞。因此，可能通过“漱口水并吐出”的接触方式而非全身给药来阻止病毒粒子的产生。
