Layne S P, Merges M J, Dembo M, Spouge J L, Conley S R, Moore J P, Raina J L, Renz H, Gelderblom H R, Nara P L
Theoretical Division, Los Alamos National Laboratory, New Mexico 87545.
Virology. 1992 Aug;189(2):695-714. doi: 10.1016/0042-6822(92)90593-e.
To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.
为了确定影响病毒灭活和中和的因素，对1型人类免疫缺陷病毒（HIV-1HXB3）的分子克隆进行了物理、化学和生物学检测。这包括定量电子显微镜检测、gp120和p24酶联免疫吸附测定、逆转录酶测定以及定量感染性测定。对于刚收获的病毒株，在每毫升含有10⁹至10¹⁰个物理颗粒的病毒储备液中，感染性病毒颗粒与非感染性病毒颗粒的比例范围为10⁻⁴至10⁻⁷。每个HIV病毒颗粒上的gp120凸起相对较少，平均每总颗粒数约为10个。每个HIV病毒颗粒平均含有约5×10⁻¹⁷克的p24和约2×10⁻¹⁶克的RNA聚合酶，分别对应约1200个和80个分子。gp120包膜蛋白从病毒粒子上的自发脱落呈指数形式，半衰期约为30小时。病毒粒子中RNA聚合酶活性的丧失也呈指数形式衰减，半衰期约为40小时。与gp120脱落和聚合酶丧失速率相比，病毒粒子的物理解体和p24核心蛋白的溶解较慢（半衰期大于100小时）。发现HIV-1感染性的衰减符合叠加的单 hit和多 hit动力学。在短预孵育时间时，感染性的丧失与gp120从病毒粒子上的自发脱落相关。在较长时间时，加速的衰减速率表明HIV感染CD4⁺细胞需要最少数量的gp120分子。重组可溶性CD4（sCD4）和膦甲酸盐（膦甲酸）的阻断活性分别随每个病毒粒子上gp120分子的数量和活性RNA聚合酶分子的数量而变化。这些结果表明病毒粒子的物理状态极大地影响感染性和中和作用。从这些发现中获得的知识将提高体外检测的可靠性，加强对野生型毒株的研究，并促进对潜在HIV治疗药物和疫苗的评估。
