An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. “Complex” retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. “Simple” retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. Importance While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. 'Complex' retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. 'Simple' retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication.

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

The road from Rous sarcoma virus to precision medicine

In 1911, more than a century ago, Peyton Rous described a curious observation, later explained by a virus named for him that led to the discovery of oncogenes, the modern era of cancer research, and the emergent field of precision medicine (1911. J. Exp. Med. https://doi.org/10.1084/jem.13.4.397).

50th anniversary of the discovery of reverse transcriptase

The simultaneous discovery in 1970 of reverse transcriptase in virions of retroviruses by Howard Temin and David Baltimore was perhaps the most dramatic scientific moment of the second half of the 20th century. Ten years previously, Temin’s observation of cells transformed by Rous Sarcoma virus led him to the conclusion that retroviruses replicate through a DNA intermediate he called the provirus. This heretical hypothesis was greeted with derision by fellow scientists; Temin and Baltimore performed a simple experiment, rapidly reproduced, and convincing to all. Its result was a major paradigm shift—reversal of the central dogma of molecular biology. It immediately grabbed the attention of both the scientific and lay press. It also came at a key time for cancer research, at the start of the “War on Cancer.” As a theoretical base and fundamental molecular tool, it enabled a decade of (largely fruitless) search for human oncogenic retroviruses but laid the foundation for the discovery of HIV 13 years later, leading to the development of effective therapy. I had the good fortune, as a student in Temin’s lab, to witness these events. I am honored to be able to share my recollection on the occasion of their 50th anniversary.

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was ∼100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.