scholarly journals HIV-1 Nef and CycK:CDK13 antagonize SERINC5 for optimal viral infectivity

2021 ◽  
Author(s):  
Qingqing Chai ◽  
Sunan Li ◽  
Morgan K. Collins ◽  
Rongrong Li ◽  
Iqbal Ahmad ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Restriction Factor ◽  
Chimeric Proteins ◽  
Viral Infectivity ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Endocytic Machinery ◽  
Affinity Purification Mass Spectrometry ◽  
Hiv 1

HIV-1 Nef antagonizes SERINC5 by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identified cyclin K and cyclin-dependent kinase 13 (CycK:CDK13) as a new Nef-associated kinase complex. CycK:CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counter activity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we created chimeric proteins between CD8 and SERINC5. Nef not only downregulates, but importantly, also binds to this chimera in a S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.

scholarly journals SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Matthew A. Szaniawski ◽  
Adam M. Spivak ◽  
James E. Cox ◽  
Jonathan L. Catrow ◽  
Timothy Hanley ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Restriction Factor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Type Iii ◽  
Type Iii Ifn ◽  
Hiv 1

ABSTRACTMacrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted.IMPORTANCEOur experimental results demonstrate that SAMHD1 dephosphorylation at threonine-592 represents a central mechanism of HIV-1 restriction that is common to the three known families of IFNs. While IFN types I and II were potent inhibitors of HIV-1, type III IFN showed modest to undetectable activity. Regulation of SAMHD1 by IFNs involved changes in phosphorylation status but not in protein levels. Phosphorylation of SAMHD1 in macrophages occurred at least in part via CDK1. Tyrosine kinase inhibitors similarly induced SAMHD1 dephosphorylation, which protects macrophages from HIV-1 in a SAMHD1-dependent manner. SAMHD1 is a critical restriction factor regulating HIV-1 infection of macrophages.

scholarly journals HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Jing Shi ◽  
Ran Xiong ◽  
Tao Zhou ◽  
Peiyi Su ◽  
Xihe Zhang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Viral Pathogenesis ◽  
Living Cells ◽  
Restriction Factor ◽  
Accessory Protein ◽  
Viral Infectivity ◽  
Bafilomycin A1 ◽  
Mhc I ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTThe primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+early, Rab7+late, and Rab11+recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCEHuman immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escapein vivothrough downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

scholarly journals Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Isabelle Staropoli ◽  
Jérémy Dufloo ◽  
Anaïs Ducher ◽  
Pierre-Henri Commere ◽  
Anna Sartori-Rupp ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cellular Proteins ◽  
Viral Infectivity ◽  
Viral Particles ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACT The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

scholarly journals Proline Residues within Spacer Peptide p1 Are Important for Human Immunodeficiency Virus Type 1 Infectivity, Protein Processing, and Genomic RNA Dimer Stability

2002 ◽  
Vol 76 (22) ◽  
pp. 11245-11253 ◽  
Author(s):  
Melissa K. Hill ◽  
Miranda Shehu-Xhilaga ◽  
Suzanne M. Crowe ◽  
Johnson Mak
Keyword(s):  
Protein Processing ◽  
Viral Infectivity ◽  
Genomic Rna ◽  
Stem Loop ◽  
Altered Protein ◽  
Immunodeficiency Virus ◽  
Rna Dimer ◽  
Hiv 1

ABSTRACT The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

scholarly journals The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Yimeng Wang ◽  
Qinghua Pan ◽  
Shilei Ding ◽  
Zhen Wang ◽  
Jingyou Yu ◽  
...  
Keyword(s):  
Envelope Proteins ◽  
Viral Resistance ◽  
Target Cells ◽  
V3 Loop ◽  
Viral Infectivity ◽  
Viral Susceptibility ◽  
Loop Sequence ◽  
Hiv 1 ◽  
Soluble Cd4

ABSTRACT Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition. IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding-induced epitopes.

scholarly journals Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

2021 ◽  
Author(s):  
Sarah E. Hickson ◽  
Eden Brekke ◽  
Johannes Schwerk ◽  
Indraneel Saluhke ◽  
Shivam Zaver ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Structural Features ◽  
Differential Sensitivity ◽  
Restriction Factor ◽  
Dependent Manner ◽  
Tetratricopeptide Repeats ◽  
Rna Interaction

ABSTRACTAlphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5’UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3’ untranslated region (3’UTR) of the virus. We investigated the potential role of variability in the 3’UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3’UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3’UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3’UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3’UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

scholarly journals The SAM domain of mouse SAMHD1 is critical for its activation and regulation

2017 ◽  
Author(s):  
Olga Buzovetsky ◽  
Chenxiang Tang ◽  
Kirsten Knecht ◽  
Jenna M. Antonucci ◽  
Li Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bound States ◽  
Restriction Factor ◽  
Activation Process ◽  
Catalytic Core ◽  
Sam Domain ◽  
And Function ◽  
Hiv 1 ◽  
Viral Reverse Transcription

ABSTRACTHuman SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular nucleotides required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory SAM domain. In this study, we determined the first crystal structure of full-length SAMHD1 by capturing mouse SAMHD1 (mSAMHD1) structures in three different nucleotide bound states. Although mSAMHD1 and hSAMHD1 are highly similar in sequence and function, we found that mSAMHD1 possesses a more complex nucleotide-induced activation process, highlighting the regulatory role of the SAM domain. Our results provide new insights into the regulation of SAMHD1 activity, thereby will facilitate the improvement of HIV mouse models and the development of new therapies for certain cancers and autoimmune diseases.

scholarly journals Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms

2021 ◽  
Author(s):  
Malene E. Lindholm ◽  
David Jimenez-Morales ◽  
Han Zhu ◽  
Kinya Seo ◽  
David Amar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Affinity Purification ◽  
Protein Protein Interaction ◽  
Cellular Hypertrophy ◽  
C Terminus ◽  
Associated Proteins ◽  
Cardiac Sarcomeres ◽  
Affinity Purification Mass Spectrometry

Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca 2+ -signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Conclusions: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

Primitive Hematopoietic Cells Resist HIV-1 Infection Via P21Waf1/Cip1/Sdi1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 490-490
Author(s):  
Jie Lin Zhang ◽  
Clyde S. Crumpacker ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Surrogate Marker ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Hematopoietic stem cells are resistant to HIV-1 infection. We have identified a novel mechanism by which the cyclin-dependent kinase inhibitor, p21Waf1/Cip1/Sdi1 (p21), known for its regulation of stem cell pool size (1,2), restricts HIV-1 infection of primitive hematopoietic cells in a non-cell cycle dependent manner. Knocking down p21 by siRNA increased HIV-1 infection and induction of p21 expression by phorbol ester (TPA) blocked HIV-1 replication. P21 did not affect the overall levels of cDNA synthesis, but significantly blocked viral integration and resulted in marked increase in 2-LTR circles, a surrogate marker of abortive integration. Consistent with these observations, p21 coimmunoprecipitated with viral integrase and both were detected in the preintegration complex (PIC). Furthermore, silencing p27Kip1 and p18INK4C, cyclin dependent kinase inhibitors related to p21 that affect cell cycle, revealed no impact on viral DNA integration. A closely related dual-tropic lentivirus with a distinct integrase, SIVmac-251 and the other cell-intrinsic inhibitors of HIV-1, Trim5a, PML, Murr1, and IFN-a were unaffected by p21. These results indicate a new function for p21, participating in prevention of HIV integration into the cellular genome. Therefore p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain the basis for these cells being an uninfected ‘sanctuary’ in HIV disease.

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