KewalRamani, V. | bioRxiv Science

bioRxiv2020

Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

HIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

microbiology↗

bioRxiv2017

Interferon-inducible miR-128 modulates HIV-1 replication by targeting TNPO3 mRNA

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three isoforms of TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also controls HIV-1 replication by repressing TNPO3. TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Manipulation of miR-128 levels in HIV target cell lines and in primary human CD4 T-cells by over-expression or knockdown showed that modulation of TNPO3 by miR-128 affects HIV-1 replication but not MLV infection. In addition, we found that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus and in cells depleted of CPSF6, suggesting that miR-128-indued TNPO3 repression is partly required for miR-128-induced inhibition of HIV-1 replication. Finally, challenging miR-modulated Jurkat cells or primary CD4 T-cells with wildtype, replication-competent HIV-1 shows that miR-128 significantly delays spreading infection. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication partly by targeting TNPO3.

cell biology↗