HIV-1 Uncoating and Nuclear Import Precede the Completion of Reverse Transcription in Cell Lines and in Primary Macrophages.

Ashwanth C Francis,Kristina Palermino-Rowland,Mariana Marin,Gregory B Melikyan,Mathew J Prellberg

doi:10.3390/v12111234

Ashwanth C Francis, Kristina Palermino-Rowland + Show 3 more

Open Access

https://doi.org/10.3390/v12111234

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Journal: Viruses	Publication Date: Oct 30, 2020
Citations: 43	License type: CC BY 4.0

Affiliation: Children's Healthcare of Atlanta, Emory University

Abstract

An assembly of capsid proteins (CA) form the mature viral core enclosing the HIV-1 ribonucleoprotein complex. Discrepant findings have been reported regarding the cellular sites and the extent of core disassembly (uncoating) in infected cells. Here, we combined single-virus imaging and time-of-drug-addition assays to elucidate the kinetic relationship between uncoating, reverse transcription, and nuclear import of HIV-1 complexes in cell lines and monocyte-derived macrophages (MDMs). By using cyclophilin A-DsRed (CDR) as a marker for CA, we show that, in contrast to TZM-bl cells, early cytoplasmic uncoating (loss of CDR) is limited in MDMs and is correlated with the efficiency of reverse transcription. However, we find that reverse transcription is dispensable for HIV-1 nuclear import, which progressed through an uncoating step at the nuclear pore. Comparison of the kinetics of nuclear import and the virus escape from inhibitors targeting distinct steps of infection, as well as direct quantification of viral DNA synthesis, revealed that reverse transcription is completed after nuclear import of HIV-1 complexes. Collectively, these results suggest that reverse transcription is dispensable for the uncoating step at the nuclear pore and that vDNA synthesis is completed in the nucleus of unrelated target cells.

Highlights

The cone-shaped mature HIV-1 core encasing the viral ribonucleoprotein complex is an assembly of ~200–250 hexamers and 12 pentamers of the capsid protein (CA) [1,2]
Uncoating of the viral core is critical for the efficient reverse transcription of the vRNA genome into a viral cDNA, which is mediated by the HIV-1 reverse transcriptase (RT) and takes place within the confines of the reverse transcription complex (RTC) [4,5]
We sought to visualize key steps of single HIV-1 infection—docking at the nuclear envelope, uncoating, and nuclear import followed by integration and reporter gene expression—and correlate these events with the acquisition of resistance to inhibitors targeting distinct steps of infection, using a bulk infectivity assay

Summary

Introduction

The cone-shaped mature HIV-1 core encasing the viral ribonucleoprotein (vRNP) complex is an assembly of ~200–250 hexamers and 12 pentamers of the capsid protein (CA) [1,2]. The conical viral core is disassembled through a poorly understood process called uncoating, which is generally defined as a partial or complete loss of CA from the conical core (reviewed in [3]). Following the completion of reverse transcription, the long-terminal repeats (LTRs) of the vDNA ends are processed by the enzyme integrase (IN) to form the pre-integration complex (PIC), which is poised for integration [6,7]. We will use the term “viral replication complexes (VRCs)” to collectively represent the vRNP, RTC, and PIC forms of HIV-1 that cannot be distinguished by imaging assays [9]

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