scholarly journals A mimic of HIV-1 nucleocapsid protein impairs reverse transcription and displays antiviral activity

1999 ◽  
Vol 96 (9) ◽  
pp. 4886-4891 ◽  
Author(s):  
S. Druillennec ◽  
C. Z. Dong ◽  
S. Escaich ◽  
N. Gresh ◽  
A. Bousseau ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Hiv 1

scholarly journals SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charlotte Martinat ◽  
Arthur Cormier ◽  
Joëlle Tobaly-Tapiero ◽  
Noé Palmic ◽  
Nicoletta Casartelli ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcription ◽  
Immune Cells ◽  
Viral Restriction ◽  
Antiviral Function ◽  
Hiv 1 ◽  
Constitutively Active

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

scholarly journals Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

2005 ◽  
Vol 89 (5) ◽  
pp. 3470-3479 ◽  
Author(s):  
Hsiao-Wei Liu ◽  
Gonzalo Cosa ◽  
Christy F. Landes ◽  
Yining Zeng ◽  
Brandie J. Kovaleski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Strand Transfer ◽  
Minus Strand ◽  
Single Molecule Fret ◽  
Transfer Step ◽  
Hiv 1

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

2008 ◽  
Vol 383 (5) ◽  
pp. 1112-1128 ◽  
Author(s):  
Sarah Bourbigot ◽  
Nick Ramalanjaona ◽  
Christian Boudier ◽  
Gilmar F.J. Salgado ◽  
Bernard P. Roques ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Primer Binding Site ◽  
Hiv 1

scholarly journals Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Jennifer A. Bohn ◽  
Justin DaSilva ◽  
Siarhei Kharytonchyk ◽  
Maria Mercedes ◽  
Jennifer Vosters ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nucleic Acid ◽  
Antiviral Activity ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Viral Dna ◽  
Dna Substrates ◽  
Apobec3 Proteins ◽  
Rna Interaction ◽  
Hiv 1

ABSTRACT APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination. IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

scholarly journals Human Immunodeficiency Virus Type 1 Central DNA Flap: Dynamic Terminal Product of Plus-Strand Displacement DNA Synthesis Catalyzed by Reverse Transcriptase Assisted by Nucleocapsid Protein

2001 ◽  
Vol 75 (7) ◽  
pp. 3301-3313 ◽  
Author(s):  
Laurence Hameau ◽  
Josette Jeusset ◽  
Sophie Lafosse ◽  
Dominique Coulaud ◽  
Etienne Delain ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Strand Displacement ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3′-5′ branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

scholarly journals p21 Restricts HIV-1 in Monocyte-Derived Dendritic Cells through the Reduction of Deoxynucleoside Triphosphate Biosynthesis and Regulation of SAMHD1 Antiviral Activity

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Jose Carlos Valle-Casuso ◽  
Awatef Allouch ◽  
Annie David ◽  
Gina M. Lenzi ◽  
Lydia Studdard ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dendritic Cells ◽  
Antiviral Activity ◽  
Immune Responses ◽  
Reverse Transcription ◽  
Kinase Inhibitor ◽  
Cell Model ◽  
Dntp Pool ◽  
Intracellular Dntp ◽  
Hiv 1

ABSTRACT HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1. IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

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