NC-mediated nucleolar localization of retroviral gag proteins

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCEHEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future. Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in many parts of the world (1, 2). Major outbreaks and epidemics are most common in developing countries, where a lack of proper sanitation conditions leads to waterborne spread of the disease. In industrialized countries, HEV infection is typically sporadic and endemic. Recently, chronic HEV infection has become an important clinical problem in immunosuppressed individual...

Section: Methodsmentioning

confidence: 99%

Section: Rps17 and Rps19 Insertions Within The Hev Hvr Contain Nls Momentioning

confidence: 99%

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

Kenney

Meng

2015

“…We find this possibility to be quite intriguing because PI(4,5)P 2 is present in the nucleus and has been implicated in mRNA processing, mRNA export, chromatin remodeling, and transcriptional regulation (50-57), whereas nuclear PI(3,4,5)P 3 has been linked to the inhibition of apoptosis through its interaction with the nucleolar protein B23 (58). Thus, we propose the hypothesis that Gag may recruit nuclear PI(4,5)P 2 or PI(3,4,5)P 3 into the Gag-genomic RNA complex in the nucleus or nucleolus, as we have recently demonstrated nucleolar trafficking of RSV Gag (47). After exiting the nucleus through the nuclear pore, PI(4,5)P 2 or PI(3,4,5)P 3 may play a role in directing the viral ribonucleoprotein complex to the PM for final assembly of the virus particle.…”

mentioning

confidence: 69%

“…5A). We previously reported that these basic residues constitute an NLS in NC (21,46,47), so we examined whether the loss of the KKRK residues would reduce the nuclear localization of Gag.NC.M1 in the presence of a point mutation in p10 that disrupts the NES. Gag.L219A-YFP (Fig.…”

mentioning

confidence: 99%

Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein

Nadaraia-Hoke

Bann

Lochmann³

et al. 2013

Self Cite

Retroviral Gag proteins direct virus particle assembly from the plasma membrane (PM). Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] plays a role in PM targeting of several retroviral Gag proteins. Here we report that depletion of intracellular PI(4,5)P 2 and phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P 3 ] levels impaired Rous sarcoma virus (RSV) Gag PM localization. Gag mutants deficient in nuclear trafficking were less sensitive to reduction of intracellular PI(4,5)P 2 and PI(3,4,5)P 3 , suggesting a possible connection between Gag nuclear trafficking and phosphoinositide-dependent PM targeting. The retroviral Gag polyprotein orchestrates the assembly of virus particles, which are released from the plasma membrane (PM) of infected cells. The major domains of Gag include matrix (MA), capsid (CA), and nucleocapsid (NC), with MA containing the PM-targeting signal (1-9). Studies to determine how MA directs membrane targeting of Gag revealed that interactions with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] and other negatively charged lipid molecules are required for proper localization of several retroviral Gag proteins, including human immunodeficiency viruses type 1 and type 2 (HIV-1 [10-13] and HIV-2 [12]), murine leukemia virus (MLV) (1,14), and Mason-Pfizer monkey virus (MPMV) (3, 7). In the case of HIV-1, Gag is directed to the PM by a bipartite signal in MA consisting of N-terminal myristic acid modification and a cluster of basic amino acids (9). Stable association of HIV-1 Gag with the PM is achieved when PI(4,5)P 2 is bound by this basic cleft in MA, leading to insertion of the myristoyl chain into the PM (10, 15). Similarly, MLV MA is myristoylated and binds PI(4,5)P 2 in the presence of phosphatidylserine but lacks an obvious PI(4,5)P 2 binding cleft (1). The MPMV MA protein must be myristoylated to bind PI(4,5)P 2 , which it binds with lower affinity than does HIV-1 or HIV-2 MA (3). In contrast, equine infectious anemia virus and human Tlymphotropic virus type 1 are less dependent than HIV-1 on specific binding of PI(4,5)P 2 for Gag recruitment to the PM and particle release (16-18).Rous sarcoma virus (RSV) Gag, which is acetylated (19, 20) but not myristoylated, undergoes nuclear trafficking prior to PM localization owing to nuclear localization signals (NLS) in the MA and NC domains. Nuclear export is mediated by a nuclear export signal (NES) in p10, an RSV-specific sequence upstream of the CA domain (21-23). The NLS in MA overlaps the membrane-binding domain (MBD), suggesting the need for an ordered sequence of trafficking events from the nucleus to the plasma membrane (8). However, the mechanism by which RSV Gag is targeted to the PM after it exits the nucleus is not well understood.The RSV Gag MBD spans the first 86 amino acids of MA and contains 11 basic residues that form a basic patch on a surfaceexposed region of the protein, similarly to other retroviral MBDs (8,24,25). The structural similarity between the RSV MBD and those of other retroviruses suggests that RSV...

“…This is supported by several pieces of evidence, including the presence of two nuclear localization signals in NC and MA recognized by different importins (21) and the detection of a large amount of RSV Gag in the nuclei of cells treated with leptomycin B (LMB) (22), a specific inhibitor of the karyopherin CRM1 (chromosome region maintenance 1 receptor) nuclear export pathway. An RSV Gag mutant that bypasses the nucleus packages vRNA less efficiently than the wild type (wt), and both nuclear trafficking and vRNA packaging is restored by the insertion of a heterologous nuclear localization signal (23,24). It has been suggested that the formation of the RSV Gag-vRNA complex induces a conformational change of Gag, which leads to the exposure of the nuclear export signal, a leucinerich region within p10 (25).…”

mentioning

confidence: 99%

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

Füzik

Píchalová

Schur

et al. 2016

The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCEAssembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential proteinprotein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV. The retroviral structural polyprotein Gag contains three conserved domains, matrix (MA), capsid (CA), and nucleocapsid (NC). Gag plays the primary role in immature particle assembly and viral genomic RNA (vRNA) recruitment and packaging.Retroviruses assemble via two different morphogenetic pathways; the first, historically referred to as C-type, wherein particle assembly occurs at the cell membrane, and the second, D-type, assembling in perinuclear regions. The pathogenic human viruses, HIV and human T-cell lymphotropic virus assemble via C-type intermediates, whereas M-PMV is the prototypic D-type retrovirus. The Gag polyprotein of M-PMV is first transported to an intracytoplasmic pericentriolar site, where particle assembly...