The goal of the research efforts in the Retrovirus Assembly Section is to extend our understanding of basic mechanisms in retroviral replication and pathogenesis. This understanding may provide new techniques or reagents that could be useful in gene therapy, and may also lead to new methods of combatting retrovirus-induced disease, including AIDS. There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein selects the viral RNA for packaging during virus assembly. This recognition involves zinc fingers in the protein. We are studying the mechanism by which the Gag protein recognizes and packages the genomic RNA of the virus during assembly in vivo. Our research strongly suggests that the recognition signal involves the three-dimensional structure formed by a dimer of genomic RNA molecules. Our mutational studies also show that the zinc fingers have other crucial functions, in addition to their role in the recognition process. These additional functions are now under investigation. Second, the Gag polyprotein and its cleavage product, the nucleocapsid (NC) protein, exhibit nucleic acid chaperone activity. That is, they transiently destabilize base pairs, catalyzing conformational transitions to the optimally base-paired structure in a nucleic acid molecule. This sequence-independent activity is used before or during virus assembly, when the Gag polyprotein promotes the annealing of a cellular tRNA molecule to the viral RNA; the tRNA is the primer for reverse transcription when the virus infects a new host cell. The activity is used again during virus maturation (i.e., after Gag is cleaved by the viral protease), when NC induces a conformational rearrangement in the viral RNA dimer within the particle. The activity also appears to be crucial during reverse transcription, facilitating both polymerization and strand-transfer steps during proviral DNA synthesis; recent data suggest that it may be important during the integration of the DNA into the host chromosome as well. We are studying the molecular mechanism underlying the nucleic acid chaperone activity of these proteins. We have also found that the HIV-1 Gag polyprotein is able to assemble into minute spherical virus-like particles in a completely defined system in vitro. The assembly requires the presence of nucleic acid, and must involve both GaguGag and Gagunucleic acid interactions. The nucleic acid requirement can be fulfilled by oligodeoxynucleotides as short as 10u15 nucleotides. The virus-like particles are only 25u30 nm in diameter, whereas the cores of authentic virions formed in mammalian cells are ~100 nm in diameter. Remarkably, if assembly reactions are performed in the presence of reticulocyte lysates, particles of 100 nm, rather than 25u30 nm, are formed. Therefore, mammalian cells contain a factor that alters the radius of curvature with which Gag polyprotein molecules interact with each other during the assembly process. This factor presumably acts during the normal assembly process in the cell; we are now trying to identify it. It is noteworthy that nucleic acid is required for assembly in vitro. This finding suggests that nucleic acid might be essential for assembly of viral particles in vivo as well. However, it has been known for many years that the genomic RNA of the virus is completely dispensable for assembly. We have recently found that RNA acts as scaffolding in retrovirus particles. In particles assembled without genomic RNA, mRNAs from the cell perform this function. These experiments have exploited alphavirus-based vectors for high-level expression of retroviral gene products. We have also analyzed the structure of immature and mature murine leukemia virus particles by cryoelectron microscopy. We found that these particles exhibit no detectable icosahedral symmetry, contrary to many statements in the literature. The immature particles contain visible subunits (presumably Gag polyprotein molecules), which are arranged with hexagonal paracrystalline packing. The mature particles lack this visible structural regularity. The absence of icosahedral symmetry implies that retrovirus particles are assembled according to different principles from those used by other spherical viruses. In addition, we have used surface plasmon resonance technology to analyze the binding of HIV-1 NC protein to very short oligonucleotides. Although NC is probably capable of binding to any single-stranded DNA or RNA, these studies showed that it exhibits profound sequence preferences. We are engaged in a detailed investigation of the binding of NC and Gag to short, well-defined oligonucleotides; this information should help us understand the various interactions with nucleic acids discussed above (i.e., chaperone activity of both NC and Gag, assembly of virus-like particles by Gag, and the exquisitely specific encapsidation of genomic RNA by Gag during virus assembly in vivo). Finally, the fact that NC facilitates reverse transcription means that it is a potential target for antiviral therapy. In fact, both murine leukemia virus and HIV-1 can be inactivated by compounds that oxidize the zinc fingers in NC. These compounds may be useful lead compounds for antiviral therapy.
| Feng, Y X; Moore, S P; Garfinkel, D J et al. (2000) The genomic RNA in Ty1 virus-like particles is dimeric. J Virol 74:10819-21 |
| Yuan, B; Campbell, S; Bacharach, E et al. (2000) Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses. J Virol 74:7250-60 |
