Analysis of CA Content and CPSF6 Dependence of Early HIV-1 Replication Complexes in SupT1-R5 Cells.

HIV-1 infects host cells by fusion at the plasma membrane, leading to cytoplasmic entry of the viral capsid encasing the genome and replication machinery. The capsid eventually needs to disassemble, but time and location of uncoating are not fully characterized and may vary depending on the host cell. To study the fate of the capsid by fluorescence and superresolution (STED) microscopy, we established an experimental system that allows discrimination of subviral structures in the cytosol from intact virions at the plasma membrane or in endosomes without genetic modification of the virus. Quantitative microscopy of infected SupT1-R5 cells revealed that the CA signal on cytosolic HIV-1 complexes corresponded to ∼50% of that found in virions at the cell surface, in agreement with dissociation of nonassembled CA molecules from entering capsids after membrane fusion. The relative amount of CA in postfusion complexes remained stable until they reached the nuclear pore complex, while subviral structures in the nucleus of infected cells lacked detectable CA. An HIV-1 variant defective in binding of the host protein cleavage and polyadenylation specificity factor 6 (CPSF6) exhibited accumulation of CA-positive subviral complexes close to the nuclear envelope without loss of infectivity; STED microscopy revealed direct association of these complexes with nuclear pores. These results support previous observations indicating capsid uncoating at the nuclear pore in infected T-cell lines. They suggest that largely intact HIV-1 capsids dock at the nuclear pore in infected SupT1-R5 cells, with CPSF6 being a facilitator of nucleoplasmic entry in this cell type, as has been observed for infected macrophages.IMPORTANCE The HIV-1 capsid performs essential functions during early viral replication and is an interesting target for novel antivirals. Thus, understanding molecular and structural details of capsid function will be important for elucidating early HIV-1 (and retroviral in general) replication in relevant target cells and may also aid antiviral development. Here, we show that HIV-1 capsids stay largely intact during transport to the nucleus of infected T cells but appear to uncoat upon entry into the nucleoplasm. These results support the hypothesis that capsids protect the HIV-1 genome from cytoplasmic defense mechanisms and target the genome toward the nucleus. A protective role of the capsid could be a paradigm that also applies to other viruses. Our findings raise the question of how reverse transcription of the HIV-1 genome is accomplished in the context of the capsid structure and whether the process is completed before the capsid is uncoated at the nuclear pore.

Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages.

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.