Mechanisms in Retroviral Replication and Pathogenesis

The goal of the research efforts in the Retroviral Assembly Section were to extend our understanding of basic mechanisms in retroviral replication and pathogenesis.  Our hope was that this understanding would lead to new methods of combatting retrovirus-induced disease, including AIDS.

We have analyzed the properties of recombinant HIV-1 Gag protein in some detail.  When nucleic acid is added to Gag, the protein assembles into virus-like particles.  This is a very nonspecific interaction: almost any nucleic acid will support assembly.  These particles are significantly smaller than authentic viral particles, but particles of the correct size are formed if inositol phosphates, as well as nucleic acid, are provided to the protein.  In the absence of nucleic acid, the protein is in monomer-dimer equilibrium, using the previously described interface within the C-terminal domain of its capsid moiety.  We generated a mutant of Gag that remains monomeric at relatively high concentrations.  We have subjected this mutant protein to a series of hydrodynamic and biophysical analyses.  These results, together with modeling studies, indicate that the protein is folded over in solution, with its N and C termini relatively close together in three-dimensional space.  Since Gag molecules are extended rods in immature virus particles, they must undergo a drastic conformational change during particle assembly.  Collaborative studies using neutron reflectivity and other techniques have now shown that Gag extends when preferred ligands are available for both its N-terminal ("matrix") domain and its C-terminal ("nucleocapsid") domain.  Interestingly, unlike HIV-1 Gag protein, the Gag protein of the gammaretrovirus Moloney murine leukemia virus is rod-shaped in solution, just as it is in immature virus particles, and does not dimerize significantly in solution.

Gag is also a nucleic acid chaperone, and participates in the annealing of a cellular tRNA molecule to the viral RNA, where the tRNA will serve as primer for viral DNA synthesis.

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 (the structural protein of the virus particle) 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.  We are studying the structure of the dimer linkage and its possible role in packaging of viral RNA.  We have found that virus particles lacking genomic RNA contain cellular mRNA molecules in place of the viral RNA; in general, these mRNA molecules are packaged nonselectively.  We developed methods enabling us to measure the affinity of recombinant Gag protein for different RNAs.  Although we could detect preferential binding of the protein to RNA containing the packaging signal, this was only possible under highly unnatural conditions, such as supraphysiological ionic strengths.  Nevertheless, this RNA supports particle assembly more efficiently than control RNAs.  We are now testing the hypothesis that genomic RNA is selectively packaged because it initiates assembly with particularly high efficiency.

The mechanism by which nucleic acid promotes particle assembly by the Gag protein is not known.  We recently reported that a very short region of Gag, termed "SP1," undergoes a conformational change when it is at high concentration.  We proposed that this region "senses" the local Gag concentration, and that the increased Gag concentration resulting from cooperative binding to nucleic acid would thus lead to a change in Gag conformation, exposing new interfaces required for particle assembly.  This hypothesis would explain the contribution of nucleic acid to particle assembly.  Our subsequent experiments have provided extensive support for this overall hypothesis.  We showed that juxtaposition of only two SP1 molecules is sufficient to induce the conformational change and additional association of these SP1 peptides.  The data suggest that SP1-SP1 association can contribute significantly to virus particle assembly and stability.  These experiments also uncovered the molecular mechanism underlying these phenomena.

We also studied the mechanism by which the restriction factor mouse APOBEC3 blocks infection by murine retroviruses.  This restriction, unlike retroviral restriction by human APOBEC3G, does not involve hypermutation of G to A in the viral genome.

We also performed a collaborative study on the HIV-1 Rev Response Element (RRE), a structure in HIV-1 RNA that is recognized by the virus-coded protein Rev in the export of unspliced viral RNAs from the nucleus.  Using small-angle X-ray scattering, our colleague Dr. Yun-Xing Wang showed that the RRE folds into an "A" shape, and that the two primary Rev binding sites are opposite each other on the legs of the A.  They are separated by a distance of 55 Å.  This distance corresponds to the length of a dimer of Rev, suggesting that this unusual topology can explain the specificity of Rev for the RRE.  We tested this hypothesis by assaying the functional capabilities of a number of RRE mutants; all of the results supported the idea that the distance between the two sites is the key to RRE function.  We continued to dissect the RRE and to explore the implications of these results for antiviral therapy.

In collaboration with Dr. Edward Gabrielson (Johns Hopkins Medical Institution), we developed sensitive assays for the presence of mouse mammary tumor virus-related sequences in human breast cancers.  However, to date we have found no evidence for these sequences in the human samples.

Many gammaretroviruses such as MLV encode an alternative form of the Gag protein called "glycogag."  Although multiple functions of this protein have been proposed, its significance in MLV replication is still not clear.  We developed reagents for the independent expression and detection of Gag and glycogag and are analyzing the functional effects of glycogag expression.  Our results showed that glycoGag has a very significant effect on the infectivity of an MLV virus particle under certain very specific conditions.  We found that with some viral envelope proteins, glycogag expression enhances the ability of MLV particles to penetrate into new target cells.  However, glycogag impairs the ability of MLV with Ebola glycoprotein to penetrate new cells.  Our data implied that all of these glycogag effects represent antagonism towards the cellular protein Serinc5.  Another retroviral “accessory protein”, the S2 protein of equine infectious anemia virus, acts very similarly to MLV glycogag, although the two proteins are completely unrelated.