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

Mowgli Holmes,Paul D Bieniasz,Fengwen Zhang,Hans-Georg Krausslich

doi:10.1371/journal.ppat.1004961

Abstract

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.

Highlights

The HIV-1 replication cycle consists of several discrete, sequentially occurring processes, involving numerous viral and host cell components
The HIV-1 replication cycle is composed of several sequential steps
While the timing of the early steps of HIV-1 replication is quite well understood, measuring the duration of later steps is complicated by the fact that asynchrony is introduced into populations of infected cells during early steps

Summary

Introduction

The HIV-1 replication cycle consists of several discrete, sequentially occurring processes, involving numerous viral and host cell components. For the early steps in HIV-1 replication, there is a reasonably good appreciation of the kinetics with which individual steps occur. The dynamics of the early steps in HIV-1 replication, entry and reverse transcription, appear to be cell type dependent, and the rather large variability in published estimates of these dynamics may be due to variation in receptor and intracellular dNTP levels. The dynamics of the various steps of the post-integration phases of the viral life cycle, e.g. the relative timing of the onset of early versus late gene expression, and the timing of particle assembly/release relative to viral gene expression are comparatively poorly documented. A challenge in determining the dynamics of HIV-1 replication is its inherent asynchrony in populations of cells, which can obscure the underlying dynamics in individual cells. Time-of-addition experiments indicate that resistance to transcription inhibitors is acquired at ~35h after infection [11, 12]

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Conclusion