Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus

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-1 HXB3). 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 gpl20 knobs per HIV particle, mean ≈ 10 when averaged over the total particle count. Each HIV particle contained a mean ≈ 5 X 10 −17 g of p24 and ≈2 × 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 ≈ 30 hr. The loss of RNA polymerase activity in virions was also exponential, with a half-life ≈ 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life > 100 hr) compared to the gpl20 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 gpl 20 molecules for efficient infection of CD4 + cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscamet) varied with the number of gpl20 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.