scholarly journals Cytoplasmic CPSF6 regulates HIV-1 capsid trafficking and infection in a cyclophilin A-dependent manner

2020 ◽  
Author(s):  
Zhou Zhong ◽  
Jiying Ning ◽  
Emerson A. Boggs ◽  
Sooin Jang ◽  
Callen Wallace ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Cyclophilin A ◽  
Higher Order ◽  
Dependent Manner ◽  
Host Proteins ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Hiv 1

SummaryHuman immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation and capsid-CPSF6 complexes co-traffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain cell-specific variations in HIV-1 capsid trafficking and uncoating.Graphical Abstract

scholarly journals Cytoplasmic CPSF6 Regulates HIV-1 Capsid Trafficking and Infection in a Cyclophilin A-Dependent Manner

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhou Zhong ◽  
Jiying Ning ◽  
Emerson A. Boggs ◽  
Sooin Jang ◽  
Callen Wallace ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Nucleus ◽  
Viral Genome ◽  
Cyclophilin A ◽  
Higher Order ◽  
Human Protein ◽  
Dependent Manner ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages. IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4+ T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis.

scholarly journals RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

1996 ◽  
Vol 16 (9) ◽  
pp. 4942-4951 ◽  
Author(s):  
B R Graveley ◽  
E S Fleming ◽  
G M Gilmartin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Structure ◽  
Critical Determinant ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
A Site ◽  
Site Recognition

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.

scholarly journals The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 77 (23) ◽  
pp. 12507-12522 ◽  
Author(s):  
Sébastien Violot ◽  
Saw See Hong ◽  
Dina Rakotobe ◽  
Caroline Petit ◽  
Bernard Gay ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dependent Manner ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

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.

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro

1992 ◽  
Vol 12 (9) ◽  
pp. 3699-3705
Author(s):  
A Valsamakis ◽  
N Schek ◽  
J C Alwine
Keyword(s):  
Human Immunodeficiency Virus ◽  
Consensus Sequence ◽  
In Vivo Studies ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Substitution Mutations ◽  
Hiv 1

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.

scholarly journals A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

2002 ◽  
Vol 76 (24) ◽  
pp. 12934-12939 ◽  
Author(s):  
Koh Fujinaga ◽  
Dan Irwin ◽  
Ran Taube ◽  
Fan Zhang ◽  
Matthias Geyer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dimensional Structure ◽  
Stable Complex ◽  
Transcriptional Elongation ◽  
Dependent Manner ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Hiv 1

ABSTRACT The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.

scholarly journals Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression

2004 ◽  
Vol 78 (13) ◽  
pp. 6846-6854 ◽  
Author(s):  
Marco A. Calzado ◽  
Rocío Sancho ◽  
Eduardo Muñoz
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Tat Protein ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3′-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.

scholarly journals Shared Usage of the Chemokine Receptor CXCR4 by Primary and Laboratory-Adapted Strains of Feline Immunodeficiency Virus

1999 ◽  
Vol 73 (5) ◽  
pp. 3661-3671 ◽  
Author(s):  
Jennifer Richardson ◽  
Gianfranco Pancino ◽  
Rastine Merat ◽  
Thierry Leste-Lasserre ◽  
Anne Moraillon ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Feline Immunodeficiency Virus ◽  
Type I ◽  
Dependent Manner ◽  
Amino Terminal ◽  
Species Origin ◽  
Chemokine Receptor Cxcr4 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1α (SDF-1α). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1α, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.

scholarly journals Escape and Compensation from Early HLA-B57-Mediated Cytotoxic T-Lymphocyte Pressure on Human Immunodeficiency Virus Type 1 Gag Alter Capsid Interactions with Cyclophilin A

2007 ◽  
Vol 81 (22) ◽  
pp. 12608-12618 ◽  
Author(s):  
Mark A. Brockman ◽  
Arne Schneidewind ◽  
Matthew Lahaie ◽  
Aaron Schmidt ◽  
Toshiyuki Miura ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Cyclophilin A ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8+ T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T242N substitution in the capsid protein is associated with upstream mutations at residues H219, I223, and M228 in the cyclophilin A (CypA)-binding loop in B57+ individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T242N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T242N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T242N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8+ T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.

scholarly journals Cellular Compartments of Human Immunodeficiency Virus Type 1 Replication In Vivo: Determination by Presence of Virion-Associated Host Proteins and Impact of Opportunistic Infection

2000 ◽  
Vol 74 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Stephen D. Lawn ◽  
Beverly D. Roberts ◽  
George E. Griffin ◽  
Thomas M. Folks ◽  
Salvatore T. Butera
Keyword(s):  
Human Immunodeficiency Virus ◽  
Opportunistic Infection ◽  
Human Immunodeficiency Virus Type ◽  
Host Proteins ◽  
Immunodeficiency Virus ◽  
Cellular Compartments ◽  
Hiv 1

ABSTRACT Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.

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