Wansley Elizabeth K, Dillon Patrick J, Gainey Maria D, Tam James, Cramer Scott D, Parks Griffith D
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1064, USA.
Virology. 2005 Apr 25;335(1):131-44. doi: 10.1016/j.virol.2005.02.004.
A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed.
一种副粘病毒SV5突变体（rSV5-P/V-CPI-）编码6种天然存在的P/V基因替换，是I型干扰素（IFN）的有效诱导剂，且在低感染复数（moi）下生长受限，这两种表型在野生型（WT）SV5中未见。在本研究中，我们比较了WT SV5和rSV5-P/V-CPI-突变体在肿瘤细胞系和正常原代细胞培养物中的IFN敏感性。我们检验了一种假设，即WT rSV5和rSV5-P/V-CPI-引发的IFN诱导差异是低moi生长和传播差异的原因。与WT SV5相反，用rSV5-P/V-CPI-以低moi感染A549肺癌细胞，在感染后24 - 48小时病毒产生达到平台期，此时分泌的IFN水平在约100至1000 U/ml之间。基因微阵列和逆转录 - 聚合酶链反应（RT-PCR）分析确定了IFN基因和IFN刺激基因，其表达因WT和P/V突变病毒感染A549细胞而增加。在存在抗IFN-β中和抗体而非抗TNF-α中和抗体的情况下，rSV5-P/V-CPI-在A549细胞中受限的低moi生长和传播得到缓解。当用IFN预处理A549或MDA-MB-435乳腺肿瘤细胞时，WT和P/V突变病毒均显示传播延迟且病毒产量降低约10倍，但感染并未消除。使用在培养中传代有限的正常原代人上皮细胞，WT rSV5和rSV5-P/V-CPI-表现出与在A549细胞中相似的高moi生长特性。然而，对这些原代细胞以及正常人肺细胞进行IFN预处理消除了突变体和WT rSV5感染的低moi传播。总之，这些数据表明，与在某些肿瘤细胞系中的生长相比，SV5在正常原代人细胞中的生长对IFN高度敏感，无论P/V基因是野生型还是突变型。这些结果提示了一种模型，其中WT SV5在正常人体细胞中的传播取决于病毒阻止IFN合成 的能力。讨论了这些结果对使用重组副粘病毒作为载体的意义。
