Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（PV）感染动物和人的上皮细胞，其中它们通常在感染部位诱导良性增殖。然而，人类生殖器病变的恶性进展与某些人乳头瘤病毒（HPV）类型（最常见的是HPV 16）之间存在很强的相关性。我们的研究涉及针对HPV和其他靶点的疫苗的开发以及PV生命周期的阐明。我们已经开发了一种简单而有效的策略，用于产生高滴度的感染性乳头瘤病毒颗粒，其包被标记质粒，下文称为假病毒体。这种方法代表了乳头瘤病毒研究的技术突破，我们已经以几种方式利用了这一发展，如下所述。假病毒生产技术正被用于探索乳头瘤病毒病毒粒子组装的基本特征。我们已经确定，双链环状基因组的双糖苷化是大小依赖性的，但令人惊讶的是序列无关的。没有乳头瘤病毒序列的质粒被有效地包装，使得这些假病毒体异常灵活的基因转移载体。我们还确定了衣壳通过有序的二硫键形成序列成熟，这导致衣壳稳定并使其对蛋白水解具有抗性。在其他基础病毒学研究中，我们检查了乳头瘤病毒感染的早期事件。利用假病毒方法，我们确定乳头瘤病毒在早期内体中脱壳，并且病毒基因组随后由L2引导至称为ND 10体或POD的亚核结构域。我们进一步确定，ND 10 s的病毒基因组的共定位是由真实的BPV 1病毒粒子感染后的病毒基因组的有效转录所必需的。其他DNA病毒在感染后早期与ND 10相互作用，但这些不涉及衣壳蛋白的相互作用导致结构破坏。因此，通常发现ND 10在病毒感染早期具有拮抗剂功能。我们的发现是第一个明确的证据，表明在某些情况下，ND 10可以促进病毒感染的建立。我们还使用假病毒来筛选感染抑制剂，这表明弗林蛋白酶（一种细胞编码的前蛋白转化酶）的抑制剂是一种有效的拮抗剂。这一观察使我们确定乳头瘤病毒感染需要弗林蛋白酶。这一要求是由于位于所有乳头瘤病毒L2小衣壳蛋白N-末端的以前未识别的共有弗林蛋白酶切割位点的弗林蛋白酶依赖性切割。对弗林蛋白酶的需要是任何病毒进入所需的病毒体蛋白的序列特异性切割的第一个例子。然而，这种功能可能是趋同进化的一个例子，因为许多细菌毒素需要弗林蛋白酶才能从内体逃逸，而弗林蛋白酶抑制剂可防止未包衣的乳头瘤病毒颗粒逃离内体。我们已经开发了一种新的乳头瘤病毒中和试验的基础上假病毒技术。这是第一个高通量乳头瘤病毒中和试验，也是第一个不受感染性衣壳可用性严重限制的试验。它是敏感的，类型特异性，并在96孔微量滴定板格式高度重现。我们希望这种检测方法能极大地促进目前由NCI、Merck和GlaxoSmithKline进行的III期VLP疫苗试验中的免疫应答监测和免疫保护相关性评估。该试验也已被用于表明，次要结构病毒蛋白，L2的N-末端，可以诱导针对广谱的乳头瘤病毒的中和抗体。

Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines against HPV and other targets and elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. This methodology represents a technical breakthrough in papillomavirus research, and we have exploited this development in several ways, as outlined below. Pseudovirus production technology is being used to explore the basic features of papillomavirus virion assembly. We have determined that encapsidation of the double stranded circular genome is size-dependent, but surprisingly sequence-independent. Plasmids with no papillomavirus sequences are efficiently packaged, making these pseudovirions exceptionally flexible gene transfer vehicles. We have also determined that the capsids mature through an ordered sequence of disulfide bond formation that results in stabilization of the capsids and makes them resistant to proteolysis. In other basic virologic studies, we examined the early events in papillomavirus infection. Taking advantage of the pseudovirus approach, we determined that papillomaviruses uncoat in early endosomes and that the viral genome is subsequently directed by L2 to subnuclear domains called ND10 bodies or PODs. We further determined that co-localization of the viral genome at ND10s is required for efficient transcription of the viral genome after infection by authentic BPV1 virions. Other DNA viruses interact with ND10s early after infections, but these interactions, which do not involve capsid proteins, lead to disruption of the structures. Therefore, it has generally been found that ND10s have an antagonist function early in viral infections. Our findings are the first clear evidence that, in some instances, ND10s can promote establishment of viral infection. We have also used the pseudoviruses to screen for inhibitors of infection, which showed that an inhibitor of furin (a cell-encoded proprotein convertase) was a potent antagonist. This observation led us to determine that papillomavirus infection requires furin. This requirement is attributable to furin-dependent cleavage of a previously unrecognized consensus furin cleavage site located in the N-terminus of the L2 minor capsid protein of all papillomaviruses. The requirement for furin is the first example of a requirement of a sequence-specific cleavage of a virion protein for entry of any virus. However, this function may be an example of convergent evolution, as many bacterial toxins require furin for their escape from the endosome, and furin inhibition prevents the uncoated papillomavirus particle from escaping the endosome. We have developed a new papillomavirus neutralization assay based on the pseudovirus technology. This is the first high throughput papillomavirus neturalization assay and the first assay that is not severely limited by the availability of infectious capsids. It is sensitive, type-specific, and highly reproducible in a 96-well microtiter plate format. We expect this assay to greatly facilitate immune response monitoring and assessment of immune correlates of protection in the phase III VLP vaccine trials currently being conducted by the NCI, Merck, and GlaxoSmithKline. The assay has also been used to show that the N-terminus of the minor structural viral protein, L2, can induce neutralizing antibodies against a broad spectrum of papillomaviruses.