Molecular Genetics of Retrovirus Replication.

Despite the availability of effective anti-HIV drug therapy, according to UNAIDS estimates, 1 [...

Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition.

Background: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription.

Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity.

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution nuclear magnetic resonance structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity.

Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities.

Background: Human APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population.

Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription.

The mature HIV-1 nucleocapsid protein (NCp7) is generated by sequential proteolytic cleavage of precursor proteins containing additional C-terminal peptides: NCp15 (NCp7-spacer peptide 2 (SP2)-p6); and NCp9 (NCp7-SP2). Here, we compare the nucleic acid chaperone activities of the three proteins, using reconstituted systems that model the annealing and elongation steps in tRNA(Lys3)-primed (-) strong-stop DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate reverse transcriptase (RT)-catalyzed DNA extension.

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein.

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism.

Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties.

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A's deaminase and nucleic acid binding activities.

NMR structure of human restriction factor APOBEC3A reveals substrate binding and enzyme specificity.

Human APOBEC3A is a single-stranded DNA cytidine deaminase that restricts viral pathogens and endogenous retrotransposons, and has a role in the innate immune response. Furthermore, its potential to act as a genomic DNA mutator has implications for a role in carcinogenesis. A deeper understanding of APOBEC3A's deaminase and nucleic acid-binding properties, which is central to its biological activities, has been limited by the lack of structural information.

Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5'-terminal genomic RNA fragments: a paradigm for RNA removal reactions in HIV-1 reverse transcription.

During (-) strong-stop DNA [(-) SSDNA] synthesis, RNase H cleavage of genomic viral RNA generates small 5'-terminal RNA fragments (14-18 nt) that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (-) SSDNA elongation, cannot occur. Here, we describe the mechanism of 5'-terminal RNA removal and the roles of HIV-1 nucleocapsid protein (NC) and RNase H cleavage in this process.

The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability.

The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146-150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype.

Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription.

The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which remodels nucleic acid structures so that the most thermodynamically stable conformations are formed. This activity is essential for virus replication and has a critical role in mediating highly specific and efficient reverse transcription. NC's function in this process depends upon three properties: (1) ability to aggregate nucleic acids; (2) moderate duplex destabilization activity; and (3) rapid on-off binding kinetics.

Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications.

The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system.

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain.

During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301, 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation.

Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs.

Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G.

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription.

Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein.

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (-) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5' end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway.

A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein.

The HIV-1 capsid (CA) protein plays an important role in virus assembly and infectivity. Previously, we showed that Ala substitutions in the N-terminal residues Trp23 and Phe40 cause a severely defective phenotype. In searching for mutations at these positions that result in a non-lethal phenotype, we identified one candidate, W23F.

Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect.

APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e.

Monomeric APOBEC3G is catalytically active and has antiviral activity.

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity.

Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity.

We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSdelta nef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5' end appeared to have less of an effect on infectivity than mutation of the 3' end of the PPT sequence.

Alteration of nucleic acid structure and stability modulates the efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid protein.

During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3'-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3'-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro.

Human immunodeficiency virus type 1 N-terminal capsid mutants containing cores with abnormally high levels of capsid protein and virtually no reverse transcriptase.

We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity.