HUMAN HERPESVIRUS 8 INFECTION OF DERMAL MICROVASCULAR ENDOTHELIAL CELLS

卡波西肉瘤（KS）是与HIV感染相关的最常见的恶性肿瘤。人疱疹病毒-8（HHV-8）1卡波西肉瘤相关疱疹病毒，在包含KS的复杂细胞病变中始终检测到。病毒基因在内皮细胞、可能的内皮来源的梭形细胞和单核细胞中表达。受感染的内皮细胞合成包括HHV-8编码的人类生长调节蛋白同源物的基因，而单核细胞似乎被有效感染。HHV-8也已在构成基于体腔的淋巴瘤（BCBL）的恶性B细胞中发现。在BCBL中，环状HHV-8基因组持续存在于潜伏感染的B细胞中，与KS衍生的细胞相反，其在体外容易生长。虽然HHV-8已在来自患者样品的内皮细胞、B细胞和单核细胞中检测到，但从多种来源分离的病毒向内皮细胞、单核细胞和单核细胞的传播是不可能的。 已经证明体外检测效率极低-仅允许通过PCR进行检测。尚未描述伴随病毒进入的事件，也未充分记录病毒在感染细胞中的持续存在与潜伏期进展一致。我们的初步工作表明，FITC-HHV-8结合人真皮微血管内皮细胞（MVDEC）。 HHV-8的附着之后是内化和转运到细胞核。在最初的24小时内，可以通过Gardella分析检测病毒游离体，然后快速线性化大多数病毒DNA，与生产性/流产感染一致。PCR和RNA印迹杂交均能检测到HHV-8的转录本，提示MVDEC的感染可能与HHV-8基因的表达有关。 比以前描述的更有效。正在进行的研究表明，一些病毒转录本存在于10天，提高了在初始裂解感染后，潜伏病毒有时可能持续存在的可能性。我们现在建议进行试点研究，以检查HHV-8在MVDEC的生物学。首先，我们将优化有效感染所需的病毒和细胞生长条件。其次，使用标准的分子/免疫学技术，我们将描绘哪些病毒基因转录本在感染后早期表达，并将确定病毒感染是否在细胞亚群中持续存在。如果是这样，将确定持续感染细胞中HHV-8表达的模式。 第三，我们将分析病毒是如何进入MVDEC的，并通过共聚焦显微镜和透射电镜来鉴定似乎涉及的结构。 第四，我们将确定哪些蛋白质参与了HHV-8通过合成单克隆抗体到纯化的病毒粒子而附着到细胞表面，从而中和感染。相反，我们将识别细胞表面蛋白质 通过产生与MVDEC结合的单克隆抗体和鉴定阻止病毒结合的抗体来连接HHV-8。这些先导实验开发的信息和试剂将为未来的研究提供基础，以描述这种新发现的疱疹病毒如何进入人类细胞并表达可能导致细胞凋亡的病毒基因产物。 永生

Kaposi's sareoma(KS)is the most common malignancy associated with HIV infection. Human herpesvirus-8(HHV-8)1 Kaposi's sarcoma-associated herpesvirus , is consistently detected in the complex cellular lesions comprising KS. Viral genes are expressed in endothelial cells, in spindle cells of probable endothelial origin and in monocytes. The infected endothelial cells synthesize genes that include HHV-8 encoded homologs of human growth regulatory proteins, whereas monocytes appears to be productively infected. HHV-8 has also been found in the malignant B cells that make up body cavity based lymphomas(BCBLs). In BCBLs, the circular HHV-8 genome persists in latently infected B cells, that in contrast to KS derived cells grow out readily in vitro. Although HHV-8 has been detected in endothelial cells, B cells and monocytes derived from patient samples, transmission of virus isolated from multiple sources to cells in vitro has proved to be extremely inefficient - permitting detection by PCR alone. The events accompanying viral entry have not been described and persistence of virus in infected cells consistent with progression to latency, has not been well documented. Our preliminary work indicates that FITC-HHV-8 binds human dermal microvascular endothelial cells (MVDEC). Attachment of HHV-8 is followed by internalization and transport to the nucleus. During the first 24 hrs viral episomes can be detected by Gardella analysis, followed by rapid linearization of most viral DNA, consistent with a productive/abortive infection. HHV-8 transcripts can be detected by PCR and by RNA blot hybridization, suggesting infection of MVDEC is more efficient than previously described. Studies in progress show some viral transcripts are present at l0 days, raising the possibility that after initial lytic infection, latent virus may sometimes persist. We now propose pilot studies to examine the biology of HHV-8 in MVDEC. First, we will optimize the conditions of virus and cell growth required for efficient infection. Second, using standard molecular/immunologic techniques, we will delineate which viral genes transcripts are expressed early after infection and will determine whether virus infection persists in a subpopulation of cells. If so, the pattern of HHV-8 expression in persistently infected cells will be determined. Third, we will analyze how virus enters MVDEC and identify the structures that appear to be involved by confocal microscopy and transmission EM. Fourth we will determine which protein(s) are involved in attachment of HHV-8 to the cell surface through synthesis of mabs to puritied virions that neutralize infection. Conversely, we will identify cell surface proteins required for attachment of HHV-8 by generating mabs to MVDEC and identifying antibodies that prevent virus binding. The information and reagents developed by these pilot experiments will provide a basis for future studies to delineate how this newly discovered herpes virus gains access to human cells and expresses viral gene products that may lead to cellular immortalization.