title: Single-Cell and Single-Cycle Analysis of HIV-1 Replication.
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 ~3 h, but matrix causes a ~6-12 h 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 48 h 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.
authors.affiliation: Columbia University, New York, New York, United States of America; Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, The Rockefeller University, New York, New York, United States of America.
authors.forename: Mowgli
authors.initials: M
authors.lastname: Holmes
authors.affiliation: Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, The Rockefeller University, New York, New York, United States of America.
authors.forename: Fengwen
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, The Rockefeller University, New York, New York, United States of America; Howard Hughes Medical Institute, New York, New York, United States of America.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Bieniasz
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D015658:HIV Infections / Q000503:physiopathology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008018:Life Cycle Stages / Q000502:physiology*; D059467:Transcriptome; D014162:Transfection; D014773:Virology / Q000379:methods*; D019065:Virus Assembly / Q000502:physiology; D014779:Virus Replication / Q000502:physiology*