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

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1234
Author(s):  
Ashwanth C. Francis ◽  
Mariana Marin ◽  
Mathew J. Prellberg ◽  
Kristina Palermino-Rowland ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Nuclear Import ◽  
Cyclophilin A ◽  
Target Cells ◽  
Nuclear Pore ◽  
Virus Escape ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1

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.

scholarly journals Identification of Capsid Mutations That Alter the Rate of HIV-1 Uncoating in Infected Cells

2014 ◽  
Vol 89 (1) ◽  
pp. 643-651 ◽  
Author(s):  
Amy E. Hulme ◽  
Z Kelley ◽  
Eneniziaogochukwu A. Okocha ◽  
Thomas J. Hope
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Wild Type ◽  
Cellular Environment ◽  
Cellular Factors ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACTAfter viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24CA) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24CAmutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24CAmutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating.IMPORTANCEThe HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24CA, which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used anin vivoassay to examine the uncoating process in HIV-1-infected cells. We determined that p24CAmutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24CAmutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

scholarly journals HIV-1 uncoats in the nucleus near sites of integration

2020 ◽  
Vol 117 (10) ◽  
pp. 5486-5493 ◽  
Author(s):  
Ryan C. Burdick ◽  
Chenglei Li ◽  
MohamedHusen Munshi ◽  
Jonathan M. O. Rawson ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Host Protein ◽  
Productive Infection ◽  
Protein Cleavage ◽  
Integration Sites ◽  
Specificity Factor ◽  
Infected Cells ◽  
Viral Dna Integration ◽  
Hiv 1

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 μm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.

scholarly journals Nuclear Import of HIV-1

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2242
Author(s):  
Qi Shen ◽  
Chunxiang Wu ◽  
Christian Freniere ◽  
Therese N. Tripler ◽  
Yong Xiong
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Integration Site ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
New Paradigm ◽  
Retroviral Infection ◽  
Dividing Cells ◽  
Pore Complexes ◽  
Hiv 1

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial–temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

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

2020 ◽  
Author(s):  
Anastasia Selyutina ◽  
Mirjana Persaud ◽  
Kyeongeun Lee ◽  
Vineet KewalRamani ◽  
Felipe Diaz-Griffero
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Viral Genome ◽  
Cellular Compartment ◽  
Nuclear Compartment ◽  
Nuclear Compartments ◽  
The Core ◽  
Biochemical Assays ◽  
Infected Cells ◽  
Hiv 1

SUMMARYHIV-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.

scholarly journals N6-methyladenosine binding proteins negatively regulate HIV-1 infectivity and viral production

2018 ◽  
Author(s):  
Wuxun Lu ◽  
Nagaraja Tirumuru ◽  
Pratibha C. Koneru ◽  
Chang Liu ◽  
Mamuka Kvaratskhelia ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Protein Translation ◽  
Leader Sequence ◽  
Target Cells ◽  
Inhibitory Effects ◽  
Domain Family ◽  
Viral Production ◽  
Infected Cells ◽  
Insight Into ◽  
Hiv 1

AbstractBackgroundThe internalN6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3, or Y1-3) bind to m6A-modified cellular mRNA and modulate its metabolism and processing, thereby affecting protein translation in cells. We previously reported that HIV-1 RNA contains m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription. Here we extended our studies to further understand the mechanisms of Y1-3-mediated inhibition of HIV-1 infection and viral production.ResultsOverexpression of Y1-3 proteins in HIV-1 target cells decreased viral genome RNA (gRNA) levels and inhibited early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5’ leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5’ leader sequence of gRNA demonstrated significantly lower infectivity compared with wild-type HIV-1, confirming that these sites are important for viral infection. HIV-1 produced from cells with knockdown of endogenous Y1, Y3, or Y1-3 proteins together showed increased viral infectivity compared with HIV-1 produced from control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag formed a complex with RNA in HIV-1-infected target cells.ConclusionsOur results suggest the inhibitory effects of Y1-3 proteins on HIV-1 infection and provide new insight into the mechanisms of m6A modification of HIV-1 RNA in regulating viral replication, which clarify some discrepancies in the previously published studies in this area.

scholarly journals Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

2009 ◽  
Vol 84 (2) ◽  
pp. 729-739 ◽  
Author(s):  
Lise Rivière ◽  
Jean-Luc Darlix ◽  
Andrea Cimarelli
Keyword(s):  
Nuclear Import ◽  
Matrix Protein ◽  
Active Phase ◽  
Cell Types ◽  
Target Cells ◽  
Nuclear Pore ◽  
Viral Dna ◽  
The Matrix ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.

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

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 620 ◽  
Author(s):  
David Bejarano ◽  
Maria Puertas ◽  
Kathleen Börner ◽  
Javier Martinez-Picado ◽  
Barbara Müller ◽  
...  
Keyword(s):  
Viral Replication ◽  
Reverse Transcription ◽  
Human Monocyte ◽  
Digital Pcr ◽  
Productive Infection ◽  
Target Cells ◽  
Post Entry ◽  
Early Replication ◽  
Kinetics Of ◽  
Hiv 1

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.

scholarly journals Cyclophilin A protects HIV-1 from restriction by human TRIM5α

2019 ◽  
Author(s):  
Kyusik Kim ◽  
Ann Dauphin ◽  
Sevnur Komurlu ◽  
Leonid Yurkovetskiy ◽  
William E. Diehl ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Cyclophilin A ◽  
Cellular Protein ◽  
Human Cells ◽  
Sub Saharan Africa ◽  
Restriction Factor ◽  
Target Cells ◽  
Chromosomal Dna ◽  
Sub Saharan ◽  
Hiv 1

The capsid (CA) protein lattice of HIV-1 and other retroviruses encases viral genomic RNA and regulates steps that are essential to retroviral invasion of target cells, including reverse transcription, nuclear trafficking, and integration of viral cDNA into host chromosomal DNA1. Cyclophilin A (CypA), the first cellular protein reported to bind HIV-1 CA2, has interacted with invading lentiviruses related to HIV-1 for millions of years3–7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8–12, but stimulates infectivity in non-human primate cells13–15. Genetic and biochemical data suggest that CypA interaction with CA protects HIV-1 from a restriction factor in human cells16–20. Discovery of the CA-specific restriction factor TRIM5α21, and of TRIM5-CypA fusion genes that were independently generated at least four times in phylogeny4,5,15,22–25, pointed to human TRIM5α as the CypA-sensitive restriction factor. However, significant HIV-1 restriction by human TRIM5α21, let alone inhibition of such activity by CypA26, has not been detected. Here, exploiting reverse genetic tools optimized for primary human CD4+T cells, macrophages, and dendritic cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to restriction by human TRIM5α, with the block occurring before reverse transcription. Identical findings were obtained with single-cycle vectors or with replication-competent HIV-1, including sexually-transmitted clones from sub-Saharan Africa. Endogenous TRIM5α was observed to associate with virion cores as they entered the macrophage cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent, but potent inhibition, imposed by human TRIM5α. Hopefully this reinvigorates development of CypA-inhibitors for treatment of HIV-1 and other CypA-dependent pathogens27–30.

scholarly journals SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A

2018 ◽  
Vol 92 (13) ◽  
pp. e00229-18 ◽  
Author(s):  
Xinlong Luo ◽  
Wei Yang ◽  
Guangxia Gao
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Leukemia Virus ◽  
Cyclophilin A ◽  
Cellular Protein ◽  
Wild Type Virus ◽  
Nuclear Pore ◽  
Nuclear Entry ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA.IMPORTANCEHIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process.

scholarly journals Continuous HIV-1 Escape from Autologous Neutralization and Development of Cross-Reactive Antibody Responses Characterizes Slow Disease Progression of Children

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 260
Author(s):  
Stefania Dispinseri ◽  
Mariangela Cavarelli ◽  
Monica Tolazzi ◽  
Anna Maria Plebani ◽  
Marianne Jansson ◽  
...  
Keyword(s):  
Disease Progression ◽  
Antibody Responses ◽  
Viral Escape ◽  
Target Cells ◽  
Transmitted Virus ◽  
Slow Progressors ◽  
Vaccine Antigens ◽  
Kinetics Of ◽  
Hiv 1

The antibodies with different effector functions evoked by Human Immunodeficiency Virus type 1 (HIV-1) transmitted from mother to child, and their role in the pathogenesis of infected children remain unresolved. So, too, the kinetics and breadth of these responses remain to be clearly defined, compared to those developing in adults. Here, we studied the kinetics of the autologous and heterologous neutralizing antibody (Nab) responses, in addition to antibody-dependent cellular cytotoxicity (ADCC), in HIV-1 infected children with different disease progression rates followed from close after birth and five years on. Autologous and heterologous neutralization were determined by Peripheral blood mononuclear cells (PBMC)- and TZMbl-based assays, and ADCC was assessed with the GranToxiLux assay. The reactivity to an immunodominant HIV-1 gp41 epitope, and childhood vaccine antigens, was assessed by ELISA. Newborns displayed antibodies directed towards the HIV-1 gp41 epitope. However, antibodies neutralizing the transmitted virus were undetectable. Nabs directed against the transmitted virus developed usually within 12 months of age in children with slow progression, but rarely in rapid progressors. Thereafter, autologous Nabs persisted throughout the follow-up of the slow progressors and induced a continuous emergence of escape variants. Heterologous cross-Nabs were detected within two years, but their subsequent increase in potency and breadth was mainly a trait of slow progressors. Analogously, titers of antibodies mediating ADCC to gp120 BaL pulsed target cells increased in slow progressors during follow-up. The kinetics of antibody responses to the immunodominant viral antigen and the vaccine antigens were sustained and independent of disease progression. Persistent autologous Nabs triggering viral escape and an increase in the breadth and potency of cross-Nabs are exclusive to HIV-1 infected slowly progressing children.

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