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The initial stages of HIV-1 infection involve the transport of the viral core into the nuclear compartment. The presence of the HIV-1 core in the nucleus triggers the translocation of CPSF6/CPSF5 from paraspeckles into nuclear speckles, forming puncta-like structures. While this phenomenon is well-documented, the efficiency of CPSF6 translocation to nuclear speckles upon HIV-1 infection varies depending on the type of cell used. In some human cell lines, only 1-2% of the cells translocate CPSF6 to nuclear speckles when exposed to a 95% infection rate. To address the issue that only 1-2% of cells translocate CPSF6 to nuclear speckles when a 95% infection rate is achieved, we screened several human cell lines and identified a human a cell line in which approximately 85% of the cells translocate CPSF6 to nuclear speckles when 95% infection rate is achieved. This cellular system has enabled the development of a robust fluorescence microscopy method to quantify the translocation of CPSF6 into nuclear speckles following HIV-1 infection. This assay holds the potential to support studies aimed at understanding the role of CPSF6 translocation to nuclear speckles in HIV-1 infection. Additionally, since the translocation of CPSF6 into nuclear speckles depends on the physical presence of the viral core in the nucleus, our method also serves as a reporter of HIV-1 nuclear import., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Overexpression of the human Sad-1-Unc-84 homology protein 2 (SUN2) blocks HIV-1 infection in a capsid-dependent manner. In agreement, we showed that overexpression of SUN1 (Sad1 and UNC-84a) also blocks HIV-1 infection in a capsid-dependent manner. SUN2 and the related protein SUN1 are transmembrane proteins located in the inner membrane of the nuclear envelope. The N-terminal domains of SUN1/2 localizes to the nucleoplasm while the C-terminal domains are localized in the nuclear lamina. Because the N-terminal domains of SUN1/2 are located in the nucleoplasm, we hypothesized that SUN1/2 might be interacting with the HIV-1 replication complex in the nucleus leading to HIV-1 inhibition. Our results demonstrated that SUN1/2 interacts with the HIV-1 capsid, and in agreement with our hypothesis, the use of N-terminal deletion mutants showed that SUN1/2 proteins bind to the viral capsid by using its N-terminal domain. SUN1/2 deletion mutants correlated restriction of HIV-1 with capsid binding. Interestingly, the ability of SUN1/2 to restrict HIV-1 also correlated with perinuclear localization of these proteins. In agreement with the notion that SUN proteins interact with the HIV-1 capsid in the nucleus, we found that restriction of HIV-1 by overexpression of SUN proteins do not block the entry of the HIV-1 core into the nucleus. Our results showed that HIV-1 restriction is mediated by the interaction of SUN1/2N-terminal domains with the HIV-1 core in the nuclear compartment., (© 2021. The Author(s).)

To understand the role of the HIV-1 capsid in viral replication, we developed a protocol to biochemically track capsid in the nucleus during infection. To this end, we separated HIV-1-infected cells into nuclear and cytosolic fractions. Fractions were analyzed by western blotting for HIV-1 capsid content as well as for nuclear and cytosolic markers to assess the bona fide origin of the fractions. This protocol can be applied in both cycling and non-cycling human cells. For complete details on the use and execution of this protocol, please refer to Selyutina et al. (2020a)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. Although a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bi-directional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement towards the nucleus.

Background: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation., Results: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1., Conclusions: We mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.

TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.

Background: TRIM5α and TRIMCyp are cytoplasmic proteins that bind incoming retroviral capsids and mediate early blocks to viral infection. TRIM5 proteins form cytoplasmic bodies, which are highly dynamic structures. So far, TRIM5 proteins have been found only in the cytoplasm of cells. Interestingly, other proteins from the TRIM family localize to the nucleus. Therefore, we tested the possibility that TRIM5 proteins traffic to the nucleus and the impact of this trafficking on retroviral restriction., Results: Here we report that the TRIM5α proteins of two Old World primates, humans and rhesus monkeys, are transported into the nucleus and are shuttled back to the cytoplasm by a leptomycin B-sensitive mechanism. In leptomycin B-treated cells, these TRIM5α proteins formed nuclear bodies that also contained TRIM19 (PML). Deletion of the amino terminus, including the linker 1 (L1) region, resulted in TRIM5α proteins that accumulated in nuclear bodies. Leptomycin B treatment of TRIM5α-expressing target cells only minimally affected the restriction of retrovirus infection., Conclusions: We discovered the ability of human and rhesus TRIM5α to shuttle into and out of the nucleus. This novel trafficking ability of TRIM5α proteins could be important for an as-yet-unknown function of TRIM5α.