The retroviruses, a large family of RNA viruses found in all eukaryotes, include important pathogens such as the human immunodeficiency viruses (HIVs). These viruses have an extraordinary lifestyle: the RNA genome is converted into a double-stranded DNA, which is inserted into the host genome and then transcribed to make new viral RNAs.
As a direct result of the integration of the viral DNA into the host genome, retroviruses can cause an impressive variety of effects in an infected host. The viruses persist in the genome for the lifetime of an infected cell and an infected individual; they can even be transmitted to offspring in the germline as integrated DNA. Many cause leukemias and other malignancies by inappropriately activating genes near the site of viral DNA insertion. They can also acquire host genes and carry them into newly infected individuals, causing a range of distinctive and aggressive tumors. These properties make the retroviruses formidable pathogens. Blocking their replication will require a deep understanding of the functions of each of the viral proteins.
Early Events of Retrovirus Infection
We are using a combination of biochemical and genetic methods to analyze the steps that occur early in infection. We have isolated mutant cell lines that are resistant to retroviral infection by both the murine leukemia viruses (MuLVs) and HIVs, and we are using DNA-mediated transfection and DNA microarrays to identify and characterize the host genes involved in these steps. Overexpression or underexpression of several host proteins (such as IQGAP, moesin, and FEZ1, which are regulators of the cytoskeleton) can profoundly block retroviral infection. Some of these cell lines exhibit abnormalities in cytoskeletal structures and intracellular trafficking of surface receptors that correlate with their virus resistance. Many genes controlling the formation of a specialized subset of microtubules—the so-called "stable" microtubules— have large effects on virus infection. We have recently found that HIV-1 induces the formation of stable microtubules soon after entry into the cell to promote its movement toward the nucleus.
Late Stages of Infection
Our laboratory is also interested in the processes of viral gene expression and the assembly of virion particles, and many efforts have been directed toward identifying host genes involved in these stages of virus replication. We have isolated dominant-acting mutant versions of several nuclear host proteins that block HIV-1 RNA formation and processing, identified components of transcriptional silencing complexes that block MuLV transcription in embryonic stem (ES) cells, and characterized a highly structured RNA suppressor of translational termination (a so-called pseudoknot) that is required for synthesis of the retroviral Pol gene products.
We have screened large mammalian cDNA expression libraries for genes that can block virus replication and have identified ZAP, a novel zinc finger protein with potent antiviral activity. Analysis of infected cells shows that overexpression of ZAP causes a profound and specific reduction in the levels of viral mRNA in the cytoplasm, without affecting levels in the nucleus. ZAP exhibits potent activity against several other RNA viruses, including alphaviruses such as Sindbis virus, suggesting that it may represent part of an innate antiviral immune response. We have found that ZAP binds specifically to these viral RNAs and utilizes the RNA exosome, a nuclease machine, to degrade them.
In a chronically infected cell, several viral proteins are brought together under the plasma membrane to form spherical particles that bud outward and are released from the cell surface. The major player in assembly is the viral Gag protein, often called "the particle-making machine." We have been particularly active in utilizing the two-hybrid system, a method for screening for protein-protein interactions between partners expressed in yeast, to identify contacts that are important in virus assembly and to select for mutants with altered binding. These studies included analysis of Gag-Gag interactions, the dimerization of the subunits of reverse transcriptase (RT), and the trimerization of the transmembrane subunits of the envelope protein.
We have also identified several novel host proteins that bind to the Moloney MuLV Gag precursor (including the endophilins, likely involved in inducing membrane curvature), to RT, to the viral integrase enzyme, and to the envelope protein. Some of these proteins are incorporated into virion particles, and dominant-negative forms of several others can act to suppress virion assembly and release.
Epigenetic Silencing of Retroviral Transcription in Embryonic Stem Cells
Retrovirus replication is strongly restricted in mouse ES and embryonic carcinoma cells at the level of transcription. Silencing of retroviruses in these pluripotent cells is likely to have evolved to protect the embryo not only from infection but also from the reactivation of endogenous retroviruses and retrotransposons that could cause damaging mutations in the germline and early progenitor cells.
Although the virus resistance of ES cells has been known for 30 years, the mechanism of transcriptional repression has been mysterious. To uncover this mechanism, we have isolated a large protein complex from ES cells and determined that it contains TRIM28 (Kap-1), a known transcriptional corepressor. Viral silencing by ES cells requires TRIM28 and is correlated with TRIM28's association with the viral DNA, as measured by chromatin immunoprecipitation. TRIM28's activity further requires a binding site for HP1, a protein implicated in silencing heterochromatin.
Additional work allowed us to identify the novel zinc finger protein ZFP809 as the ES cell–specific recognition molecule that binds viral DNA and brings TRIM28 to the integrated provirus. We found that expression of ZFP809 is sufficient to render even differentiated cells highly resistant to MuLV infection. We suggest that ZFP809 evolved as a stem cell–specific retroviral restriction factor and therefore constitutes a novel component of the intrinsic immune system of stem cells. We have recently identified new players in the silencing complex (YY1; EBP1) and are studying the regulation of expression of these factors in ES cells.
Grants from the National Institutes of Health supported some of the studies on murine retrovirus replication and silencing.
As of March 1, 2016