Single-Cell and Single-Cycle Analysis of HIV-1 Replication

The dynamics of the late stages of the hiv-1 life cycle are poorly documented. viral replication dynamics are typically measured in populations of infected cells,but asynchrony that is introduced during the early steps of hiv-1 replication complicates the measurement of the progression of subsequent steps and can mask replication dynamics and their variation in individual infected cells. we established microscopy-based methods to dynamically measure hiv-1-encoded reporter gene and antiviral gene expression in individual infected cells. we coupled these measurements with conventional analyses to quantify delays in the hiv-1 replication cycle imposed by the biphasic nature of hiv-1 gene expression and by the assembly-inhibiting property of the matrix domain of gag. we further related the dynamics of restriction factor (apobec3g) removal to the dynamics of hiv-1 replication in individual cells. these studies provide a timeline for key events in the hiv-1 replication cycle,and reveal that the interval between the onset of early and late hiv-1 gene expression is only ~3h,but matrix causes a ~6–12h delay in the generation of extracellular virions. interestingly,matrix delays particle assembly to a time at which apobec3g has largely been removed from the cell. thus,a need to prepare infected cells to be efficient producers of infectious hiv-1 may provide an impetus for programmed delays in hiv-1 virion genesis. our findings also emphasize the significant heterogeneity in the length of the hiv-1 replication cycle in homogenous cell populations and suggest that a typical infected cell generates new virions for only a few hours at the end of a 48h lifespan. therefore,small changes in the lifespan of infected cells might have a large effect on viral yield in a single cycle and the overall clinical course in infected individuals. © 2015 holmes et al.