ROCK1 and LIM Kinase Modulate Retrovirus Particle Release and Cell-Cell Transmission Events

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing fulllength viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNAdependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5= packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.KEYWORDS Gag, MS2, packaging, Psi, RNA trafficking, RRE, genomes, human immunodeficiency virus, translation, virus assembly T he spatial distribution of mRNAs within the cytoplasm is a core determinant of mRNA turnover, cytoplasmic utilization, and the formation of functional macromolecular complexes (1-3). Viruses face severe challenges in this regard during the p...

Section: Resultsmentioning

confidence: 99%

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

Becker

Sherer

2017

“…In herpes simplex virus 1 (HSV-1)-infected neuronal cells, the level of phosphorylated cofilin was found to decrease at an early stage of infection (ϳ2 h after infection) and then to continuously increase during the later stages of infection, causing biphasic F-actin dynamics (48). Knockdown of cofilin reduced virus production but did not affect protein expression in cells infected with HSV-1 or retroviruses (48,49). In contrast, MV RNA synthesis was suppressed in cofilin knockdown cells, presumably because of its involvement in the MV RNP complex formation.…”

Section: Discussionmentioning

confidence: 99%

Actin-Modulating Protein Cofilin Is Involved in the Formation of Measles Virus Ribonucleoprotein Complex at the Perinuclear Region

Koga

Sugita

Noda

et al. 2015

In measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and the nucleocapsid (N) protein, phosphoprotein (P protein), and large protein, assembles at the perinuclear region and synthesizes viral RNAs. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of formation of the RNP complex results in the reduction of viral RNA synthesis. Cofilin phosphorylation on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and the phosphorylated form of cofilin is preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing formation of the RNP complex and viral RNA synthesis. IMPORTANCE Many Measles is a highly contagious viral disease characterized by high fever, respiratory symptoms, conjunctivitis, and a macropapular rash. The patients exhibit immunosuppression, often resulting in secondary infections, the main cause of measles-associated mortality and morbidity. On rare occasions, measles virus (MV) persists in the central nervous system and causes subacute sclerosing panencephalitis with long incubation periods (1). Although mortality and morbidity have been greatly reduced thanks to worldwide vaccination efforts, the World Health Organization estimates that 145,700 people died of measles in 2013 (http://www .who.int/mediacentre/factsheets/fs286/en/).MV, a member of the genus Morbillivirus in the family Paramyxoviridae, possesses 6 genes coding for the nucleocapsid (N) protein, phosphoprotein (P protein), matrix (M) protein, and hemagglutinin (HA), fusion, and large proteins. The two envelope glycoproteins, the hemagglutinin and fusion proteins, mediate receptor binding and membrane fusion, respectively (1). Pathogenic MVs use as cellular receptors the signaling lymphocyte activation molecule (SLAM) (2, 3) and nectin 4 (4, 5), which are expressed on immune and epithelial cells, respectively. The N, P, and large proteins, together with the viral genome, constitute the ribonucleoprotein (RNP) complex, which is found at the perinuclear region in MV-infected cells (6-8) and functions as the RNAdependent RNA polymerase. The M protein is involved in virion assembly and the budding process (1).Besides the P protein, the MV P gene encodes two additional proteins, C and V (9, 10). The V mRNA is produced through the insertion of a single nontemplated guanine at the editing site of the P gene during the transcription. The V protein possesses 231 amino acid residues in the N terminus which are identical to those possessed by the P protein and 68 unique resid...

“…In addition, small interfering RNA (siRNA) depletion of Filamin A, a host cell protein that regulates actin network dynamics, impairs viral particle assembly and release (39). Other cell effectors that regulate actin turnover, such as LIMK1 and ROCK1, were recently described to play a role in retrovirus release (40). Altogether, these results tend to show, directly or indirectly, a role for the regulation of the cortical actin cytoskeleton during HIV assembly and/or release, especially in CD4 T cells.…”

mentioning

confidence: 99%

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Thomas

Mariani-Floderer

López-Huertas

et al. 2015

During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCEDuring HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.