Search Results (31)

The continued effective control of retroviral infections will no doubt require the development of new clinical interventions targeting underexploited areas of retroviral biology such as genome selection and virion assembly. In our previous work, we demonstrated that both the Gag-psi (Ψ) interaction and genomic RNA (gRNA) dimerization each uniquely contribute to the formation, morphology, and stability of Rous sarcoma virus (RSV) Gag-viral RNA (vRNA) biomolecular condensates (BMCs). The present work builds upon those observations, utilizing atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) to elucidate the nanoscale morphology, resistance to mechanical deformation, and constituent diffusivity of RSV Gag-vRNA BMCs. These approaches revealed a novel role for gRNA dimerization in nanoscale condensate architecture and mechanical stability that aids in our understanding of why gRNA dimerization is critical for efficient packaging of the retroviral genome. Further biophysical characterization of RSV Gag-gRNA BMCs therefore possesses great potential to reveal novel avenues for therapeutic intervention. Full article

Murine leukemia viruses (MuLVs) are retroviruses that cause various diseases in mice and have served as a model for retrovirus replication and pathogenesis. MuLVs are separated into infectious exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs) that are remnants of ancient infectious XRVs. Detection of XRVs in the original host cells has some difficulties because of the high similarity in sequence between ERVs and XRVs and expression of some ERV genes. There are some techniques available for monitoring retroviral replication, but each comes with limitations in terms of labor intensity, detection range, cost, and phases after infection. This study developed a novel quantitative PCR (qPCR) method for assessing replication of Moloney MuLV (M-MuLV) in mouse cells. The method amplified the region in packaging signal and gag and distinguished exogenous M-MuLV from ERVs with mouse SC-1 cells. The qPCR system could quantify viral sequences in infected cells from 16 to 72 h post-infection, with a 3-log range of difference. This was compared with the traditional infectivity assay, focal immunofluorescence assay. In conclusion, the developed qPCR system provides a rapid, sensitive, and scalable alternative for quantifying M-MuLV infectivity, with potential for broader applications in MuLV research. Full article

Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization. We observed that the psi (Ψ) packaging signal and the dimerization initiation sequence (DIS) had stabilizing effects on RSV Gag condensates, while RNAs lacking these features promoted or antagonized condensation, depending on local protein concentration and condensate architecture. An RNA containing Ψ, DIS, and the dimerization linkage structure (DLS) that is capable of stable dimer formation was observed to act as a bridge between RSV Gag condensates. These observations suggest additional, condensate-related roles for Gag-Ψ binding, gRNA dimerization, and Gag dimerization/multimerization in gRNA selection and packaging, representing a significant step forward in our understanding of how these interactions collectively facilitate efficient genome packaging. Full article

A fundamental idea for targeting glioblastoma cells is to exploit the neurotropic properties of Zika virus (ZIKV) through its two outer envelope proteins, prM and E. This study aimed to develop envelope glycoproteins for pseudotyping retroviral vectors that can be used for efficient tumor cell infection. Firstly, the retroviral vector pNLlucAM was packaged using wild-type ZIKV E to generate an E-HIVluc pseudotype. E-HIVluc infection rates for tumor cells were higher than those of normal prME pseudotyped particles and the traditionally used vesicular stomatitis virus G (VSV-G) pseudotypes, indicating that protein E alone was sufficient for the formation of infectious pseudotyped particles. Secondly, two envelope chimeras, E41.1 and E41.2, with the E wild-type transmembrane domain replaced by the gp41 transmembrane and cytoplasmic domains, were constructed; pNLlucAM or pNLgfpAM packaged with E41.1 or E41.2 constructs showed infectivity for tumor cells, with the highest rates observed for E41.2. This envelope construct can be used not only as a tool to further develop oncolytic pseudotyped viruses for therapy, but also as a new research tool to study changes in tumor cells after the transfer of genes that might have therapeutic potential. Full article

This review is an accompaniment to a Special Issue on “Retroviral RNA Processing”. It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay. Full article

Two non-covalently linked copies of the retrovirus genome are specifically recruited to the site of virus particle assembly and packaged into released particles. Retroviral RNA packaging requires RNA export of the unspliced genomic RNA from the nucleus, translocation of the genome to virus assembly sites, and specific interaction with Gag, the main viral structural protein. While some aspects of the RNA packaging process are understood, many others remain poorly understood. In this review, we provide an update on recent advancements in understanding the mechanism of RNA packaging for retroviruses that cause disease in humans, i.e., HIV-1, HIV-2, and HTLV-1, as well as advances in the understanding of the details of genomic RNA nuclear export, genome translocation to virus assembly sites, and genomic RNA dimerization. Full article

Integrase is the retroviral protein responsible for integrating reverse transcripts into cellular genomes. Co-packaged with viral RNA and reverse transcriptase into capsid-encased viral cores, human immunodeficiency virus 1 (HIV-1) integrase has long been implicated in reverse transcription and virion maturation. However, the underlying mechanisms of integrase in these non-catalytic-related viral replication steps have remained elusive. Recent results have shown that integrase binds genomic RNA in virions, and that mutational or pharmacological disruption of integrase-RNA binding yields eccentric virion particles with ribonucleoprotein complexes situated outside of the capsid shell. Such viruses are defective for reverse transcription due to preferential loss of integrase and viral RNA from infected target cells. Parallel research has revealed defective integrase-RNA binding and eccentric particle formation as common features of class II integrase mutant viruses, a phenotypic grouping of viruses that display defects at steps beyond integration. In light of these new findings, we propose three new subclasses of class II mutant viruses (a, b, and c), all of which are defective for integrase-RNA binding and particle morphogenesis, but differ based on distinct underlying mechanisms exhibited by the associated integrase mutant proteins. We also assess how these findings inform the role of integrase in HIV-1 particle maturation. Full article

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC. Full article

Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses. Full article

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo. Full article

Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55^Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55^Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55^Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle. Full article

The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus. Full article

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR. Full article

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions. Full article