Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses

Abstract

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

Figure 1. General structures of the viruses used in the recombination and packaging assays.

H0-RRE is shown on top and elements of other viruses different from H0-RRE are labelled. Two marker genes are located in nef of each virus; the translation of the gfp gene was directed by IRES. The inactivating mutations in gfp are shown as asterisks. Black box represents the HIV-1 RRE, whereas gray box represents four copies of MPMV CTE.

Figure 2. The fusion-dependent recombination assay.

(A) An example of the fusion experiment, with predicted outcomes, is shown schematically using the H0-PTAP^–-infected 293T.CC cell line (293T.CC.H0-PTAP^–) and a T6-NC*-infected 293T cell line (293T.T6-NC*). The reciprocal experiment using 293T.CC.T6-NC* and 293T.H0-PTAP^– was also performed. (B) Representative flow cytometry analyses of target cells infected with viruses harvested from various fusion experiments. The cells used in the fusion experiments, from which viruses were harvested, are indicated above each flow cytometry plot; all 293T cells were transiently transfected with plasmid expressing HIV-1 Env. The x- and y-axis show Thy and HSA expression, respectively. Panel I, mock infection; panel V, positive control, viruses harvested from a producer cell line dual-infected with H0-PTAP^– and T6-NC*. Panels II, III, VI, and VII, viruses harvested from fusing one cell line containing a mutant provirus with another cell line that does not contain any HIV-1 provirus; panels IV and VIII, viruses harvested from fusing a T6-NC*-containing cell line with an H0-PTAP^–-containing cell line. (C) Observed recombination rates from the fusion-based recombination assay with (in gray) or without (in white) the drug roscovitine. Recombination rates generated using viruses harvested from dual-infected cells are shown for comparison. Means and standard deviations generated from three experiments are shown.

Figure 3. Effect of nuclear export pathway on recombination.

(A) Titers of the HSA and Thy, or HSA and B7 viruses generated from producer cell lines. White bars represent titers of viruses encoding HSA, whereas black bars represent titers of viruses encoding Thy or B7, respectively. (B) Observed recombination rates using viruses from each cell line. Means and standard deviations generated from three experiments are shown.

Figure 4. General structures of the plasmids used for single virion visualization.

(A) Modified HIV-1 genomes used in the study. Abbreviations are the same as Fig. 1. Stem loop sequences recognized by BglG and MS2 coat proteins are shown in black and grey, respectively; 18 Bgl stem loops and 24 MS2 stem loops are used in these constructs although fewer stem loops are shown. (B) RNA binding proteins tagged with fluorescent proteins used in the study. Pro, Pol II promoter, NLS, nuclear localization signal, grey circle, transcription termination signal.

Figure 5. Effect of nuclear export pathway used by gag mRNA on genomic RNA packaging and recombination.

(A) Schematic representations of the expected outcomes when Gag is provided in trans by mRNA using the CRM-1 pathway. (B) Proportion of the two viruses released from the H0-CTE-Spe.T6-RRE-Spe cell line when Gag is provided in trans by mRNA using either the CRM-1 or NXF1 pathway, or both. The virus proportions when Gag is provided in cis are also shown for comparison. (C) The recombination rate between the two viruses when Gag is provided in trans. Means and standard deviation generated from three experiments are shown.