scholarly journals Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus PassageIn Vivo

2016 ◽  
Vol 90 (15) ◽  
pp. 6918-6935 ◽  
Author(s):  
Akatsuki Saito ◽  
Matthew S. Henning ◽  
Erik Serrao ◽  
Brittany N. Dubose ◽  
Samantha Teng ◽  
...  
Keyword(s):  
Life Cycle ◽  
Integration Site ◽  
Humanized Mice ◽  
Site Preference ◽  
Primary Cells ◽  
Wild Type ◽  
Nuclear Entry ◽  
Fitness Advantage ◽  
Hiv 1

ABSTRACTCleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6in vivosuggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells andin vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains.IMPORTANCECPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replicationin vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replicationin vivobut provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1in vivo.

scholarly journals TRIM5α restriction of HIV-1-N74D viruses in lymphocytes is caused by a loss of cyclophilin A protection

2020 ◽  
Author(s):  
Anastasia Selyutina ◽  
Lacy M. Simons ◽  
Angel Bulnes-Ramos ◽  
Judd F. Hultquist ◽  
Felipe Diaz-Griffero
Keyword(s):  
T Cells ◽  
Integration Site ◽  
Cyclophilin A ◽  
Site Preference ◽  
Primary Cells ◽  
Protein Cleavage ◽  
Wild Type ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Hiv 1

ABSTRACTThe core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein cleavage and polyadenylation specificity factor subunit 6 (CPSF6). In addition, HIV-1-N74D viruses have altered patterns of integration site preference in human cell lines. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit infectivity defects when compared to wild type. The reason for this loss of infectivity in primary cells is unknown. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5αhu) restriction in CD4+ T cells, we tested whether TRIM5αhu was involved in the decreased infectivity observed for HIV-1-N74D. Depletion of TRIM5αhu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5αhu. Accordingly, TRIM5αhu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5αhu restriction activity.

scholarly journals Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA

2020 ◽  
Vol 16 (12) ◽  
pp. e1009147
Author(s):  
Shelby Winans ◽  
Stephen P. Goff
Keyword(s):  
Life Cycle ◽  
Histone Modifications ◽  
Reverse Transcription ◽  
Integration Site ◽  
Viral Dna ◽  
Large Defect ◽  
Central Function ◽  
Site Distribution ◽  
Hiv 1

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.

scholarly journals In Vivo Blockade of the PD-1 Receptor Suppresses HIV-1 Viral Loads and Improves CD4+T Cell Levels in Humanized Mice

2012 ◽  
Vol 190 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Brent E. Palmer ◽  
C. Preston Neff ◽  
Jonathan LeCureux ◽  
Angelica Ehler ◽  
Michelle DSouza ◽  
...  
Keyword(s):  
T Cell ◽  
Cd4 T Cell ◽  
Humanized Mice ◽  
Viral Loads ◽  
Hiv 1

scholarly journals Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain

2005 ◽  
Vol 79 (3) ◽  
pp. 1470-1479 ◽  
Author(s):  
Isabel Scholz ◽  
Brian Arvidson ◽  
Doug Huseby ◽  
Eric Barklis
Keyword(s):  
Human Immunodeficiency Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Order Structures ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.

scholarly journals Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

2000 ◽  
Vol 74 (21) ◽  
pp. 9895-9902 ◽  
Author(s):  
Jean-Claude Twizere ◽  
Pierre Kerkhofs ◽  
Arsène Burny ◽  
Daniel Portetelle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Phosphorylation Sites ◽  
Primary Cells ◽  
Wild Type

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

scholarly journals Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

1999 ◽  
Vol 73 (10) ◽  
pp. 8831-8836 ◽  
Author(s):  
Hongmei Liu ◽  
Xiaoyun Wu ◽  
Hongling Xiao ◽  
John C. Kappes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Wild Type Virus ◽  
Strand Transfer ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

scholarly journals HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

2021 ◽  
Author(s):  
Anabel Guedán ◽  
Callum D Donaldson ◽  
Ophélie Cosnefroy ◽  
Ian A Taylor ◽  
Kate N. Bishop
Keyword(s):  
Reverse Transcription ◽  
Integration Site ◽  
Productive Infection ◽  
Nuclear Pore ◽  
Virus Infectivity ◽  
Nuclear Speckles ◽  
Nuclear Entry ◽  
The Core ◽  
Fold Reduction ◽  
Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

scholarly journals Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity

mBio ◽  
2021 ◽  
Author(s):  
Benjamin S. Goldberg ◽  
Chengzi I. Kaku ◽  
Jérémy Dufloo ◽  
Timothée Bruel ◽  
Olivier Schwartz ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Loss Of Function ◽  
Wild Type ◽  
Suboptimal Outcome ◽  
Antiviral Activities ◽  
Broadly Neutralizing Antibody ◽  
Hiv 1 ◽  
Prevention Of Hiv

Given the suboptimal outcome of VRC01 antibody-mediated prevention of HIV-1 infection in its first field trial, means to improve diverse antiviral activities in vivo have renewed importance. This work revisits a loss-of-function experiment that investigated the mechanism of action of b12, a similar antibody, and finds that the reason why complement-mediated antiviral activities were not observed to contribute to protection may be the inherent lack of activity of wild-type b12, raising the prospect that this mechanism may contribute in the context of other HIV-specific antibodies.

scholarly journals Longitudinal tracking reveals sustained polyclonal repopulation of human-HSPC in humanized mice despite vector integration bias

2020 ◽  
Author(s):  
Gajendra W. Suryawanshi ◽  
Hubert Arokium ◽  
Sanggu Kim ◽  
Wannisa Khamaikawin ◽  
Samantha Lin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Biology ◽  
Integration Site ◽  
Fetal Liver ◽  
Small Animal ◽  
Humanized Mice ◽  
Vector Integration ◽  
Longitudinal Tracking ◽  
Stem And Progenitor Cells

AbstractClonal repopulation of human hemopoietic stem and progenitor cells (HSPC) in humanized mouse models remains only partially understood due to the lack of a quantitative clonal tracking technique for low sample volumes. Here, we present a low-volume vector integration site sequencing (LoVIS-Seq) assay that requires a mere 25μl mouse blood for quantitative clonal tracking of HSPC. Using LoVIS-Seq, we longitudinally tracked 897 VIS clones—providing a first-ever demonstration of clonal dynamics of both therapeutic and control vector-modified human cell populations simultaneously repopulating in humanized mice. Polyclonal repopulation of human cells became stable at 19 weeks post-transplant indicating faster clonal repopulation than observed in humans. Multi-omics data of human fetal liver HSPC revealed that in vivo repopulating clones have significant vector integration bias for H3K36me3-enriched regions. Despite this bias the repopulation remains normal, underscoring the safety of gene therapy vectors. LoVIS-Seq provides an efficient tool for exploring gene therapy and stem cell biology in small-animal models.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2020 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Genetic Background ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Resistant Virus ◽  
Wild Type ◽  
Fitness Advantage

AbstractFavipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

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