Rous Sarcoma Research Articles

Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP) and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly.

The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

Antitumoral gene-based strategy involving nitric oxide synthase type III overexpression in hepatocellular carcinoma.

Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoblastoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. The first-generation adenoviruses were designed to overexpress NOS-3 or green fluorescent protein, and luciferase complementary DNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenovirus and Hepa 1-6 cells were used for in vitro and in vivo experiments. Adenoviruses were administered through the tail vein 2 weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8, -9 and -3 activities in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by Nω-nitro-l-arginine methyl ester hydrochloride, p53 and CD95 small interfering RNA. AFP-NOS-3/RSV-luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

Discovering the first tyrosine kinase

In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month.

Atomic Modeling of an Immature Retroviral Lattice Using Molecular Dynamics and Mutagenesis

Defining the molecular interaction between Gag proteins in an assembled hexagonal lattice of immature retrovirus particles is crucial for elucidating themechanisms of virus assembly and maturation. Recent advances in cryo-electron microscopy have yielded subnanometer structural information on themorphology of immature Gag lattices, making computational modeling and simulations feasible for investigating the Gag-Gag interactions at the atomic level. We have examined the structure of Rous sarcoma virus (RSV) using all-atom molecular dynamics simulations and invitro assembly, to create the first all-atom model of an immature retroviral lattice. Microseconds-long replica exchange molecular dynamics simulation of the spacer peptide (SP)-nucleocapsid (NC) subdomains results in a six-helix bundle with amphipathic properties. The resulting model of the RSV Gag lattice shows features and dynamics of the capsid protein with implications for the maturation process, and confirms the stabilizing role of the upstream and downstream regions of Gag, namely p10 and SP-NC.

Development of Rous sarcoma Virus-like Particles Displaying hCC49 scFv for Specific Targeted Drug Delivery to Human Colon Carcinoma Cells

Virus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug. RSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells. FITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs. These results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

Editorial: Role of lipids in virus assembly.

Editorial: Role of lipids in virus assembly.

The beginning of RNA.

In October 1993, Reinhard Luhrmann organized a small RNA meeting in Marburg, Germany, and invited me as a speaker. This was the same year the Nobel Prize for Splicing was awarded, and many of the meeting talks concerned the mechanism of splicing. I was excited that Joan Steitz, who had first proposed that snRNPs were involved in splicing, was the Keynote Speaker at this conference. She had also recently been named the President-Elect of the newly formed RNA Society. Two interacting low abundance snRNPs, U11 and U12, had been discovered the year before in the Steitz lab, but their function was unknown. My lab had recently discovered that the U11 snRNA binds to a Rous sarcoma virus (RSV) RNA element that helps regulate the balance between spliced and unspliced viral RNA. This was before AT-AC introns were discovered, and U11 and U12 snRNPs were found to substitute for U1 and U2 snRNPs in a minor class of spliceosome. I flew to Frankfurt from Baltimore, found the train station and then had to figure out how to buy a train ticket from a non-English speaking ticket machine. Finally, I arrived in Marburg, a bit jet-lagged, and went to the meeting hotel. Tim Nilsen, whom I barely knew, was sitting in the lobby when I checked in. He immediately attacked me for a critical review that he thought I had written six years earlier of his paper on m6A methylation of influenza RNA. It is possible I did write the offending review, but his paper was published nevertheless. That was when I learned that Tim never forgets a thing. I thought the meeting had a great mix of people working on mRNA splicing and on viral RNAs. Other invited speakers from the US included Bob Krug and Mike Malim. Bob had made the amazing discovery that influenza virus stole caps from cellular mRNAs. Mike studied Rev-mediated export of unspliced HIV RNA. Incidentally, Bob had also identified m6A in the influenza genomic RNA in the '70s, when my group had begun to study it in RSV. Tim talked about the role of U6 snRNP in cis and trans splicing in nematodes. Reinhard talked about his lab's detailed analysis of snRNPs and of the spliceosome. The meeting was very stimulating and interactive, ending with a dinner and dance party at Reinhard's home. Marburg turned out to be a charming small German town with a castle, despite its infamy as the site of an outbreak of Marburg virus (a relative of Ebola). I remember walking around town a lot. On one of these walks back to the hotel, Joan shared her frustration with Tim and me that the journal Cell did not appreciate RNA work. This conversation led to the idea that a new journal should be started that focused on RNA. By the end of the Marburg meeting, Tim had agreed to start such a journal and to be Editor in Chief. He asked me to be the Editor for Viral RNA. I don't think I believed he would pull it off, so I didn't leap at the chance. I had no idea it would become the leading journal for the RNA community and regret that I didn't accept his offer. To my surprise, the first copy of RNA appeared less than a year and a half later, in March 1995, as the journal for the RNA Society. It has always been both rigorous and fun to read. It has an important role as a place to get fast and fair reviews of papers on any aspect of RNA biology. I recommend it to both my graduate students and colleagues as a reasonable place to send a paper that will be seen by the RNA community. The timing of its beginning was propitious, as there has been an explosion of new findings in the RNA field in the last 20 years. I have been fascinated with the discoveries of new types of RNAs, including microRNAs and long noncoding RNAs. Genome sequencing revealed there were fewer protein-coding genes than expected, requiring massive alternative splicing to synthesize mRNAs for all the missing proteins. Mechanisms of nonsense-mediated mRNA decay (NMD) remain interesting and controversial. Finally, the m6A field seems to be undergoing a rebirth with many functions ascribed to cellular mRNAs containing this internal modification that was originally studied in viral RNAs decades ago. It is hard to imagine the RNA field without the RNA journal.

Structural Comparison of Membrane-Bound Retroviral Gag Proteins

he Gag polyprotein is essential for the assembly of retroviral particles. It is expressed in the cellular cytoplasm and targets the surface of the plasma membrane, where assembly occurs. All Gag proteins from various retroviral species contain structured domains - the membrane-binding matrix (MA), the capsid (CA) and the genome-binding nucleocapsid (NC) domains - that are separated by unstructured linker regions. Cryo-EM of immature virions shows assemblies on the plasma membrane in which Gag polyproteins are in an extended conformation, apparently stabilized by lateral interactions between their CA domains. However, the structural dynamics are complex. In a recent study with neutron reflection (NR), we showed that in HIV-1 Gag, the MA-CA linker is so flexible that the polyprotein binds a model membrane in a backfolded structure in the absence of nucleic acid, and that a transformation into an extended conformation is triggered upon nucleic acid binding (Datta et al., J. Mol. Biol. 406, 2011, 205). Here we investigate the membrane-binding of murine leukemia virus (MLV) and Rous sarcoma virus (RSV) Gag proteins, which also include unstructured linkers between their MA and CA domains. Both wild-type MLV and RSV Gag bind membranes in vitro in folded conformations, as shown by NR, indicating structural flexibility of their MA-CA linkers. In contrast, an MLV mutant, Δp12, in which the MA-CA linker has been eliminated, binds the membrane in an extended conformation. An investigation of the RSV mutant, Δ104-220, that is analogous to MLV-Δp12, is currently in progress. These studies suggest that the disordered linker regions in retroviral Gag proteins may function to prevent unproductive assembly processes in the host cytosol or on the membrane in the absence of viral genome.

Development of Tools to Study Retroviral Gag Assembly on Giant Unilamellar Vesicles(GUV)

The retroviral Gag polyprotein provides the principal driving force for virus assembly and budding from the cellular plasma membrane. The binding of Gag to the plasma membrane (PM) is governed by several mechanisms, including electrostatics, hydrophobics, Gag multimierization, and recognition of specific lipid head groups. To better understand how Gag interacts with the PM, control proteins GFP-poly(K/R)n (n=4/8/12 residues), were purified and used to compare Gag membrane binding to an electrostatic membrane binding protein. Preliminary protein-liposome binding experiments suggest that the anionic lipids, PS and PI(4,5)P2, contribute to the recruitment of polycationic proteins as expected. Unlike previous results, which showed that HIV-1 Gag responded strongly to cholesterol, GFP-poly(K/R)n responded weakly to cholesterol. PI(4,5)P2 enhances more liposome binding for retroviral Gag than for the control protein GFP-poly(K/R)n. This study may shed light on how the retroviral Gag protein interacts electrostatically with membranes, and recognizes specific lipids such as PI(4,5)P2.Membrane binding is not a prerequisite for Gag multimerization, however, it might enhance the formation of Gag-Gag interactions. It is known that assembly of Gag occurs on the PM, but it remains unclear when and where Gag multimerization takes place, and if membrane lipid composition influences Gag assembly. To visualize by fluorescence microscopy the interaction of Gag with membranes and to detect Gag-Gag interactions, Rous Sarcoma Virus Gag is labeled with different Alexa-flurophores using Sfp synthase. Giant unilamellar vesicles (GUVs), composed of lipids similar to cellular inner leaflet lipid composition, were employed to study Gag assembly by measuring fluorescence resonance energy transfer (FRET). This study may provide a starting point for understanding where Gag multimerization happens as well as how membrane composition affects Gag assembly.

Cell Association in Rous Sarcoma Virus (RSV) Rescue and Cell Infection

In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.

Membrane binding and subcellular localization of retroviral Gag proteins are differentially regulated by MA interactions with phosphatidylinositol-(4,5)-bisphosphate and RNA.

ABSTRACTThe matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P2. To examine whether PI(4,5)P2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P2 dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P2 independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P2 dependence to alleviate the membrane binding block imposed by RNA.

The Viral Etiology of Skin Cancer

The concept that viruses may be etiological agents of cancers is as old as the discovery of viruses themselves. In 1908, 3 years before Peyton Rous was passaging what later became to be known as Rous Sarcoma Virus in chickens, two Danish scientists, Ellerman and Bang, were characterizing a transmissible filtrate that reproducibly caused leukemia in chickens. These findings were received with harsh skepticism, and the scientific community did not universally accept the concept that tumors could be caused by transmissible agents. Richard Shope, a colleague of Peyton Rous at the Rockefeller Institute, identified an infectious agent that infected cottontail rabbits. It caused cutaneous papillomas that could grow to be quite large and which may be the basis of sightings of the mystical and ravenous ‘‘Jackelope’’ of southwestern American lore. Shope later collaborated with Rous to demonstrate that exposure of these papillomas to coal tar or infection of a host that does not support viral replication caused malignant progression to skin cancers. This infectious agent, the cottontail rabbit papillomavirus or Sylvilagus floridanus Papillomavirus 1, was the first virus linked to a cancer in a mammalian host (Javier and Butel, 2008; Moore and Chang, 2010) (Figure 1).

Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles.

The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability.

Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.

In Vitro Assembly of Retroviruses.

Assembly, part of the late stages of the retroviral life cycle, begins when the structural polyprotein Gag associates with viral genomic RNA. Ultimately, more than a thousand Gag molecules form a spherical immature virion. Maturation takes place soon after or concomitantly with virus budding and is initiated as Gag is cleaved by the retroviral protease into its constituent protein domains. The immature core is thought to disassemble and the liberated CA proteins to reassemble into a morphologically distinct mature capsid. In vitro assembly with derivatives of Gag and CA has been used to study retroviruses for over two decades. In this review, we examine the discovery and development of three major model systems [human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Mason-Pfizer monkey virus (MPMV)] and discuss structural features and aspects of the retroviral assembly pathway that have been uncovered using in vitro assembly. We also put forward two major unresolved questions in the field and propose future avenues of research.

Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.

SarahElizabeth Stewart.

This is a photograph of Sarah Elizabeth Stewart, PhD, MD (1905–1976) (Figure), whose discoveries involving the murine polyomavirus with Bernice Eddy, PhD, propelled the then-reluctant field of oncology to pursue viral etiologies of cancer. Sarah Stewart was born on August 16, 1905, in Jalisco, Mexico, to an American engineer and Mexican mother. After moving with her family back to the United States at age 5, Stewart remained fluent in Spanish. In 1927, she graduated from New Mexico State University and in 1930, she earned an MS in Microbiology at the University of Massachusetts, Amherst. She worked at the National Institutes of Health (NIH) during 1935–1944, publishing 7 papers on anaerobic bacteria and completing a PhD from the University of Chicago in 1939. Figure Sarah Elizabeth Stewart (1905–1976). Source: Hobson-Huntsinger University Archives at New Mexico State University. In 1944, when Stewart requested support to study the link between animal tumors and viruses, the directors of the NIH Laboratory of Microbiology and the National Cancer Institute (NCI) refused on the grounds that the proposal seemed dubious and that she lacked appropriate qualifications. Rather than give up her passion to study viruses and cancer, Stewart resigned and became a bacteriology instructor at Georgetown University School of Medicine; there she audited classes, and after women were permitted to enroll, Stewart became their first woman graduate at age 39. After completing an internship in 1951, Stewart returned to NCI to launch her viral oncology research career. To understand Stewart’s role in catalyzing viral oncology research, it is necessary to recognize that until the 1950s, scientists dismissed the idea that viruses could cause cancer. It took many decades before the seminal contributions of several virologists studying cancers were appreciated, such as Peyton Rous’ 1911 discovery of the Rous sarcoma virus (which caused tumors in chickens), and discoveries of Richard Shope (rabbit fibroma) and John Bittner (mouse mammary carcinoma) in the 1930s. In 1951, Ludwig Gross described the transmission of leukemia in newborn mice by using a cell-free extract; in 1953, he reported parotid tumors in these mice. Even after Stewart confirmed Gross’s findings in 1953, the scientific community still did not acknowledge viral causes of mammalian cancer. Only after Stewart fulfilled Koch’s postulates in 1957, with the assistance of Dr. Bernice Eddy, did oncologists pay heed to viruses. Dr. Eddy had also trained as a PhD bacteriologist at the University of Chicago and since 1937, worked at the NIH Biologics Control Laboratories, in Bethesda, Maryland. In 1954, Eddy had been sidelined for whistleblowing about the presence of live virus in Jonas Salk’s inactivated polio vaccine (the infamous Cutter incident). So in 1956, when Stewart approached Eddy for assistance growing the agent causing parotid tumors in mice, Eddy readily agreed and the 2 women rapidly worked out the characteristics of the agent that was not referred to as a virus in their publications until 1959. Together they showed that the virus produced 20 types of mouse tumors and could cause tumors in other small mammals. At Eddy’s suggestion, the virus was dubbed polyoma, which means many tumors, and they named it the SE (Stewart–Eddy) polyomavirus. They also demonstrated that the virus causes cell necrosis and proliferation in cell culture, that it is highly antigenic, and that it leads to formation of specific antibodies in infected animals whether or not tumors develop. The results of their collaboration were picked up by a 1959 Time Magazine cover story, citing John Heller, then the NCI director, “the hottest thing in cancer research is research on viruses as possible causes of cancer.” Alan Rabson, a prominent member of the NCI Laboratory of Pathology, stated, “The whole place just exploded after Sarah found polyoma.” Stewart became medical director of the NCI Laboratory of Oncology and spent the remainder of her life researching several oncogenic viruses (e.g., Epstein-Barr virus). As a US Public Health Service Commissioned Officer, her scientific contributions to the study of viral etiologies of cancer earned her the Federal Women’s Award, presented by President Lyndon Johnson in 1965. In 1960, Eddy again found herself in hot water, this time for reporting her discovery of an oncogenic simian virus (SV40) in polio vaccine prepared from monkey kidneys. Stewart retired from the Public Health Service in 1970 to become a full professor of pathology at Georgetown University. She died of stomach cancer in 1976. Bernice Eddy described her as “a forceful individual who did not let anything stand in [her] way if she could help it.” Despite sex discrimination and a period in which several laboratory directors disparaged her wish to study oncogenic viruses, Stewart persisted with such enthusiasm that she managed to break through as one of the most influential scientists and cancer researchers of her time.

Membrane interaction of retroviral Gag proteins.

Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

During virion maturation, the Rous sarcoma virus (RSV) capsid protein is cleaved from the Gag protein as the proteolytic intermediate CA-SP. Further trimming at two C-terminal sites removes the spacer peptide (SP), producing the mature capsid proteins CA and CA-S. Abundant genetic and structural evidence shows that the SP plays a critical role in stabilizing hexameric Gag interactions that form immature particles. Freeing of CA-SP from Gag breaks immature interfaces and initiates the formation of mature capsids. The transient persistence of CA-SP in maturing virions and the identification of second-site mutations in SP that restore infectivity to maturation-defective mutant viruses led us to hypothesize that SP may play an important role in promoting the assembly of mature capsids. This study presents a biophysical and biochemical characterization of CA-SP and its assembly behavior. Our results confirm cryo-electron microscopy (cryo-EM) structures reported previously by Keller et al. (J. Virol. 87:13655-13664, 2013, doi:10.1128/JVI.01408-13) showing that monomeric CA-SP is fully capable of assembling into capsid-like structures identical to those formed by CA. Furthermore, SP confers aggressive assembly kinetics, which is suggestive of higher-affinity CA-SP interactions than observed with either of the mature capsid proteins. This aggressive assembly is largely independent of the SP amino acid sequence, but the formation of well-ordered particles is sensitive to the presence of the N-terminal β-hairpin. Additionally, CA-SP can nucleate the assembly of CA and CA-S. These results suggest a model in which CA-SP, once separated from the Gag lattice, can actively promote the interactions that form mature capsids and provide a nucleation point for mature capsid assembly. The spacer peptide is a documented target for antiretroviral therapy. This study examines the biochemical and biophysical properties of CA-SP, an intermediate form of the retrovirus capsid protein. The results demonstrate a previously unrecognized activity of SP in promoting capsid assembly during maturation.