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The goal of our research is to define the molecular mechanisms involved in the replication of HIV and related retroviruses. These studies are critical for developing new strategies to combat the AIDS epidemic, which continues to spread to all parts of the world. In the absence of an effective vaccine, AIDS patients are currently being treated with a combination of anti-viral agents. The long path to drug discovery often begins with the development of a well-defined, in vitro
system that mimics in vivo
events in virus replication. Thus, we have developed reconstituted model systems to investigate the individual steps in HIV-1 reverse transcription, a major target of HIV therapy. Much of our work is focused on the viral nucleocapsid protein (NC), an essential cofactor for efficient and specific viral DNA synthesis. More recently, studies have been initiated to investigate the biochemical properties and antiviral activity of a host deaminase protein, APOBEC3G (APO3G), which is a potent inhibitor of HIV-1 infection by virions lacking the Vif protein. Other efforts in the laboratory are directed toward understanding the function of the viral capsid protein (CA) in HIV-1 assembly and early postentry events, during the course of virus replication in vivo
1. Role of Nucleocapsid Protein in HIV-1 Reverse Transcription.
HIV-1 NC is a small, basic, nucleic acid binding protein having two zinc fingers, each containing invariant CCHC zinc-coordinating residues.
Figure. 1. Schematic representation of the HIV-1 NC protein. Note the two zinc fingers and the invariant CCHC zinc-coordinating residues in each finger. The amino acid sequences of the N- and C-terminal fingers are not identical and both fingers are required for virus replication.
NC has an unusual biochemical property: it is a nucleic acid chaperone, which means that it can catalyze nucleic acid conformational rearrangements that lead to formation of the most thermodynamically stable structure. We have reported that the nucleic acid chaperone activity of NC plays a critical role in the initiation event and in the plus- and minus-strand transfer steps that occur during reverse transcription (Fig. 2). During minus-strand transfer, the first product of reverse transcription, (-) strong-stop DNA, is translocated to the 3' end of viral RNA, i.e., acceptor RNA, in a reaction mediated by base pairing of the complementary repeat regions at the 3' ends of the RNA and DNA molecules molecules (Fig. 2, step 3). We have shown that NC stimulates HIV-1 minus-strand transfer and inhibits a competing self-priming reaction induced by the highly structured TAR DNA stem-loop in (-) strong-stop DNA.
Figure 3.. Minus-strand transfer and the competing self-priming reaction. In our standard reconstituted assay system, (-) strong-stop DNA [(-) SSDNA], is provided as a synthetic 128-nt oligonucleotide, containing R (97 nt) and a portion of U5. The acceptor is a T7 transcript of 148 nt, containing R (94 nt) and a portion of U3. When NC is added, strand transfer occurs and (-) SSDNA is elongated. In the absence of NC, self-priming from the 3’ end of (-) SSDNA predominates; the 5’ overhang in (-) SSDNA acts as the template. Self-priming leads to non-productive synthesis of dead-end products, termed “SP DNAs”.
Thus, in the presence of acceptor RNA, NC nucleic acid chaperone activity destabilizes the TAR DNA structure and facilitates formation of the more stable RNA-DNA duplex in the annealing reaction. Using a mutational approach, we have also found that the native zinc fingers are required to inhibit self-priming and promote annealing. This finding demonstrates the critical importance of the zinc fingers for NC interaction with complex nucleic acid structures.
In current work we are focusing on the influence of nucleic acid secondary structure on NC nucleic acid chaperone activity. More specifically, we are investigating the structural and thermodynamic requirements for NC interaction with RNA and DNA minus-strand transfer intermediates. A series of synthetic (-) strong-stop DNA and acceptor RNA truncation mutants were constructed and have been studied by in vitro assay of minus-strand transfer and self-priming, enzymatic structure probing, and analysis of secondary structure using RNA and DNA structure prediction algorithms. As might be expected, truncations that disrupt the TAR DNA structure in (-) strong-stop DNA completely eliminate DNA self-priming. However, reducing or eliminating self-priming does not necessarily result in an increase in strand transfer efficiency: the structure of the acceptor RNA is also important. In fact, we have demonstrated that NC can only mediate efficient strand transfer when both (-) strong-stop DNA and acceptor RNA are moderately structured. Collectively, the results demonstrate that a delicate thermodynamic balance between (-) strong-stop DNA and acceptor RNA must be maintained for efficient minus-strand transfer. In current work, we are continuing to investigate the mechanism of nucleic acid chaperone activity using biochemical assays and mutational analysis.
2. Molecular analysis of catalytically active, highly purified APOBEC3G and mechanism of anti-HIV-1 activity.
APO3G is a member of a large family of cellular cytidine deaminases, which includes APO1 and AID (activation-induced cytidine deaminase). These proteins convert cytosine residues to uracil in DNA and/or RNA and they all contain at least one zinc finger domain with the conserved HECC motif.
Figure 4. Schematic diagram showing the domain structure of APO3G. For comparison, the structures of APO1 and AID are also included. APO3G has two zinc finger domains with the conserved motifs H-A-E-X23-28-P-C-X-X-C (HECC). The Glu residue is thought to serve as a proton shuttle in the deamination reaction, whereas the His and Cys residues coordinate zinc. Each zinc finger domain is followed by a linker and a pseudocatalytic domain.
APO3G has two zinc finger domains and it exhibits potent anti-HIV-1 activity in the absence of the viral protein Vif. The enzyme is also known to block reverse transcription. Until now, studies of APO3G have been performed primarily in cell-based virus replication systems and with enzyme from viral lysates. For the first time, we have obtained enzymatically active, highly purified APO3G, expressed in a baculovirus system. Our goal is to provide a detailed analysis of the biochemical properties of APO3G and to identify biochemical determinants of antiviral activity.
APO3G deaminates cytosine residues in ssDNA, but not in ssRNA, dsDNA, dsRNA, or a DNA/RNA hybrid. In contrast, electrophoretic mobility shift assays (EMSA) show that APO3G binds efficiently to ssDNA or ssRNA (Kd, 76 nM), less efficiently to a DNA/RNA hybrid, and only very poorly to dsDNA or dsRNA. These data indicate that the substrate specificities for nucleic acid binding and deamination are not correlated. To examine the individual roles of the zinc finger domains, three zinc finger mutants were expressed and purified: C100S, zinc finger 1; C291S, zinc finger 2; and the double mutant. (Note that it is the last Cys in each motif that was changed to Ser; Fig. 4.) EMSA data show that both zinc fingers bind nucleic acids, but zinc finger 1 contributes more to binding activity than zinc finger 2. Interestingly, the in vitro binding capacities of the zinc finger mutants are consistent with the extent of APO3G encapsidation into virions. In the deamination assay, C100S has wild-type activity, whereas C291S and C100S/C291S have no detectable activity. This demonstrates that the second zinc finger domain is solely responsible for deaminase activity. Moreover, data from single-round replication assays show that C100S retains significant antiviral activity, whereas C291S and the double mutant do not. These findings suggest a correlation between deamination and antiviral activity. In summary, both zinc fingers are required for nucleic acid binding and encapsidation, while zinc finger 1 is dispensable for deamination and is less important for antiviral activity than zinc finger 2. Experiments to elucidate the mechanism(s) by which APO3G inhibits HIV-1 reverse transcription are currently underway.
3. Function of HIV-1 capsid protein in virus assembly and early postentry events.
In the mature virion, HIV-1 CA forms a shell surrounding the virus core, which contains a ribonucleoprotein complex consisting of genomic RNA, the tRNA primer, and a number of viral proteins. Following virus entry, the CA protein is released from cores, allowing the ribonucleoprotein complex to initiate reverse transcription. This process is known as "uncoating" or "disassembly". Our laboratory has been investigating the role of the HIV-1 CA in virus assembly and early postentry events, a stage in the infectious process that is still not completely understood. Structural studies of CA showed that a group of conserved hydrophobic residues (including Trp23 and Phe40) faces the interior of the coiled coil-like structure within the N-terminal domain and it was suggested that these residues might be important for maintaining CA structure and function.
Figure. 5. Ribbon representation showing a front view of the N-terminal core domain of HIV-1 CA. The β-hairpin structure, the cyclophilin A (CypA) binding loop, and seven α-helices are labeled. Three residues that have been the subject of mutational analysis, Trp23, Phe40, and Asp51, are located in helices I, II, and III, respectively. (Figure from Tang et al. (2001) J. Virol. 75:9357-9366).
In an initial study, we reported the unusual phenotype associated with single alanine substitution mutations in these residues, using genetic, molecular, and ultrastructural approaches. Mutant virions are not infectious and lack the characteristic cone-shaped core. Moreover, despite having a functional reverse transcriptase (RT) enzyme, the mutants are blocked in initiation of viral DNA synthesis in infected cells. These findings suggested that the mutations result in a defect in an early step preceding reverse transcription, which is correlated with a defect in assembly of virus cores.
More recently, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. To investigate the possibility that the mutants might be compromised in an early postentry step, we modeled disassembly in vitro, by generating viral cores from particles treated with mild detergent. In general, mutant cores exhibit a normal protein profile. However, there are two striking differences from the wild-type pattern: mutant cores display a marked deficiency in RT protein and enzymatic activity and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.
To determine whether substitutions other than alanine result in a different phenotype, we have constructed a series of vertical mutations in residues Trp23 and Phe40. All of these mutants were tested in a single-cycle infectivity assay and only one mutant, W23F, exhibited infectivity, albeit at a very low level. We have recently isolated second-site suppressors of the W23F mutation and experiments to characterize the suppressor phenotype(s) are now in progress. These studies should provide additional insights into CA structure and function.