Genetic drift of HIV populations in culture

Abstract

Populations of Human Immunodeficiency Virus type 1 (HIV-1) undergo a surprisingly large amount of genetic drift in infected patients despite very large population sizes, which are predicted to be mostly deterministic. Several models have been proposed to explain this phenomenon, but all of them implicitly assume that the process of virus replication itself does not contribute to genetic drift. We developed an assay to measure the amount of genetic drift for HIV populations replicating in cell culture. The assay relies on creation of HIV populations of known size and measurements of variation in frequency of a neutral allele. Using this assay, we show that HIV undergoes approximately ten times more genetic drift than would be expected from its population size, which we defined as the number of infected cells in the culture. We showed that a large portion of the increase in genetic drift is due to non-synchronous infection of target cells. When infections are synchronized, genetic drift for the virus is only 3-fold higher than expected from its population size. Thus, the stochastic nature of biological processes involved in viral replication contributes to increased genetic drift in HIV populations. We propose that appreciation of these effects will allow better understanding of the evolutionary forces acting on HIV in infected patients.

Figure 1

A. Difference in length of the insertion in vpr gene of Vpr-FS and Vpr-FS-StuI clones allows measurements of their relative abundance in mixtures. Fragments of genomes containing insertions are amplified in RT-PCR reactions using fluorescently labeled primers. Relative abundance of products of different length can be quantitated from fluorescence intensity of the corresponding bands in GeneScan assay. B. Scheme of the experimental approach used to correlate the number of infected cells to the amount of genetic drift. Viral variants Vpr-FS and Vpr-FS-StuI are mixed in 1∶1 ratio. The mixture is serially diluted and used to infect multiple replicates of cell cultures (shown 6 replicates for each dilution). Cultures are scored as positive (black circles) or negative (white circles) for HIV infection. Cell-free virus is collected from virus-positive wells and the frequency of virions of each variant is measured by the GeneScan assay. The data is used to calculate the amount of genetic drift for each dilution. Dilutions containing positive and negative wells are used to calculate TCID50, which provides the measure of viral population size at the start of the experiment.

Figure 2. Representative results of an experiment measuring genetic drift in HIV populations.

A. The frequency of Vpr-FS in replicate cultures. Each point represents one of 12 independent cultures done for each dilution. B. Variance in frequency of Vpr-FS-StuI was calculated from data shown in panel A. Each point corresponds to average variance in frequency of Vpr-FS-StuI from initial 50%, adjusted for contribution from assay variability (see Materials and Methods). The expected variance for an ideal population is shown as straight dashed line.

Figure 3. Relationship between virus population size and genetic drift in a variety of cell cultures.

A. In C8166 cells. B. In CEMx174 cells. C. In 1∶1 mixture of C8166 and CEMx174 cells. D. In PBMCs. Each point corresponds to average variance in frequency of Vpr-FS-StuI from initial 50% (see Materials and Methods). Error bars are 95% confidence intervals. Population size N is defined as the average number of infected cells in each culture at the beginning of the experiment. The expected variance for an ideal population is shown as straight dashed line.

Figure 4. Synchronization of infection reduces genetic drift.

A. Virus mixture was first incubated with Raji-DC-SIGN cells. After one hour the cells were washed to remove unbound virus and mixed with C8166 target cells. B. Raji-DC-SIGN cells were mixed with the target C8166 cells and infected. C. Infections were synchronized. Virus mixture was incubated with target cells for 1 hour, after which the cells were washed to remove unbound virus and resuspended in fresh media. D. Infection was synchronized as in panel C. After 15 hours viral replication was blocked by addition of nevirapine. Cell-free virus was collected 48 hours later, cells were washed to remove nevirapine and resuspended in fresh media to allow viral replication and TCID50 measurement. Each point corresponds to average variance in frequency of Vpr-FS-StuI from initial 50%. Population size N is defined as the average number of infected cells in each culture at the beginning of the experiment. Error bars are 95% confidence intervals. The expected variance for an ideal population is shown as straight dashed line.