scholarly journals Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1090
Author(s):  
Nabeel Alzahrani ◽  
Ming-Jhan Wu ◽  
Saravanabalaji Shanmugam ◽  
MinKyung Yi
Keyword(s):  
Virus Assembly ◽  
Signal Peptidase ◽  
Structural Protein ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
Virus Family ◽  
Viral Structural Protein ◽  
Replication Scheme ◽  
Viral Polyprotein

The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.

scholarly journals Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

2009 ◽  
Vol 83 (17) ◽  
pp. 8379-8395 ◽  
Author(s):  
Tung Phan ◽  
Rudolf K. F. Beran ◽  
Christopher Peters ◽  
Ivo C. Lorenz ◽  
Brett D. Lindenbach
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Rna Binding ◽  
Virus Assembly ◽  
Genetic Selection ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
Enzyme Complexes

ABSTRACT The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

scholarly journals Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Claudia Del Vecchio ◽  
Michele Celestino ◽  
Marta Celegato ◽  
Giorgio Palù ◽  
Cristina Parolin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Feline Immunodeficiency Virus ◽  
Virus Assembly ◽  
Cellular Protein ◽  
Structural Protein ◽  
Virus Budding ◽  
Particle Assembly ◽  
Gag Protein ◽  
Double Mutation ◽  
Immunodeficiency Virus

ABSTRACT The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains—the matrix (MA), capsid (CA), and nucleocapsid (NC) domains—drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding. IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.

scholarly journals Impact of Capsid Anchor Length and Sequential Processing on the Assembly and Infectivity of Dengue Virus

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 32 ◽  
Author(s):  
Oscar R. Burrone ◽  
José L. Slon Campos ◽  
Monica Poggianella ◽  
Jyoti Rana
Keyword(s):  
Dengue Virus ◽  
Signal Peptidase ◽  
Sequence Length ◽  
Processing Efficiency ◽  
Particle Assembly ◽  
Luminal Side ◽  
Cytosolic Side ◽  
Experimental Approaches ◽  
Viral Polyprotein ◽  
Ns3 Protease

: The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavage events mediated by both viral and host proteases. Among these, sequential cleavage at the N- and C-termini of the hydrophobic capsid anchor (Ca) at the junction of C-PrM has been considered essential for the production of flaviviruses. Here, using a refined dengue pseudovirus production system, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and the assembly of viral particles. The replacement of the relatively short DENV2 Ca with the homologous regions from West Nile or Zika viruses or, alternatively, the increase in its length, improved cleavage, and hence particle assembly. Furthermore, we show that the substitution of the Ca conserved proline residue (Pro-110), as alanine abolishes pseudovirus production, regardless of the Ca sequence length. Using two experimental approaches, we investigated the need for sequential cleavage (first on the cytosolic side, then on the luminal side) and found that, while cleavage at the Ca-Pr boundary is essential for the assembly of infective particles, the same is not true for cleavage at the C-Ca boundary. We show that both the mature (C) and unprocessed capsids (C-Ca) of DENV2 were equally efficient in packaging the viral RNA and in assembling the infective particles. This was further confirmed with mutants, in which cleavage at the luminal side, by the signal peptidase, occurred independently of cleavage at the cytosolic side, by the viral NS2B/NS3 protease. We thus demonstrate that, unlike other flaviviruses, DENV2 capsid does not require a cleavable Ca sequence and that sequential cleavage is not an obligatory requirement for the morphogenesis of infective particles.

scholarly journals The Roles of Pol and Env in the Assembly Pathway of Human Foamy Virus

1998 ◽  
Vol 72 (5) ◽  
pp. 3658-3665 ◽  
Author(s):  
David N. Baldwin ◽  
Maxine L. Linial
Keyword(s):  
Virus Assembly ◽  
Foamy Virus ◽  
Surface Glycoprotein ◽  
Human Foamy Virus ◽  
Structural Protein ◽  
Genome Packaging ◽  
Particle Assembly ◽  
P Protein ◽  
Assembly Pathway ◽  
B Virus

ABSTRACT Human foamy virus (HFV) is the prototype of theSpumavirus genus of retroviruses. These viruses have a genomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol protein independently of their structural proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly and genome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structural protein. The Pol protein is not required for capsid assembly, and the Env surface glycoprotein is not required for release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNA requires core structural protein and polymerase (P protein) for assembly of nucleocapsids and requires surface glycoproteins for release from the cell. We investigated the requirements for synthesis of extracellular HFV particles by constructing mutants with either the pol or envgene deleted. We found that the Pol protein is dispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env, intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virus assembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.

scholarly journals Dynamic Association between HIV-1 Gag and Membrane Domains

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Ian B. Hogue ◽  
G. Nicholas Llewellyn ◽  
Akira Ono
Keyword(s):  
Plasma Membrane ◽  
Virus Assembly ◽  
Active Role ◽  
Spherical Particles ◽  
Cell Junction ◽  
Membrane Microdomains ◽  
Virological Synapse ◽  
Structural Protein ◽  
Particle Assembly ◽  
Hiv 1

HIV-1 particle assembly is driven by the structural protein Gag. Gag binds to and multimerizes on the inner leaflet of the plasma membrane, eventually resulting in formation of spherical particles. During virus spread among T cells, Gag accumulates to the plasma membrane domain that, together with target cell membrane, forms a cell junction known as the virological synapse. While Gag association with plasma membrane microdomains has been implicated in virus assembly and cell-to-cell transmission, recent studies suggest that, rather than merely accumulating to pre-existing microdomains, Gag plays an active role in reorganizing the microdomains via its multimerization activity. In this paper, we will discuss this emerging view of Gag microdomain interactions. Relationships between Gag multimerization and microdomain association will be further discussed in the context of Gag localization to T-cell uropods and virological synapses.

scholarly journals Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 216
Author(s):  
Alanna C. Tseng ◽  
Vivek R. Nerurkar ◽  
Kabi R. Neupane ◽  
Helmut Kae ◽  
Pakieli H. Kaufusi
Keyword(s):  
West Nile Virus ◽  
Enzymatic Activity ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
West Nile ◽  
Biochemical Assays ◽  
In Trans ◽  
Viral Polyprotein ◽  
Ns3 Protease

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

scholarly journals Identification of a Structural Element in HIV-1 Gag Required for Virus Particle Assembly and Maturation

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Mariia Novikova ◽  
Lucas J. Adams ◽  
Juan Fontana ◽  
Anna T. Gres ◽  
Muthukumar Balasubramaniam ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Critical Role ◽  
Replication Cycle ◽  
Structural Defects ◽  
Fold Axis ◽  
Structural Element ◽  
Particle Assembly ◽  
Compensatory Mutations ◽  
Hiv 1 ◽  
Selection Of

ABSTRACTLate in the HIV-1 replication cycle, the viral structural protein Gag is targeted to virus assembly sites at the plasma membrane of infected cells. The capsid (CA) domain of Gag plays a critical role in the formation of the hexameric Gag lattice in the immature virion, and, during particle release, CA is cleaved from the Gag precursor by the viral protease and forms the conical core of the mature virion. A highly conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122 to 125) [PPIP(122–125)] in a loop connecting CA helices 6 and 7 resides at a 3-fold axis formed by neighboring hexamers in the immature Gag lattice. In this study, we characterized the role of this PPIP(122–125) loop in HIV-1 assembly and maturation. While mutations P123A and P125A were relatively well tolerated, mutation of P122 and I124 significantly impaired virus release, caused Gag processing defects, and abolished infectivity. X-ray crystallography indicated that the P122A and I124A mutations induce subtle changes in the structure of the mature CA lattice which were permissive forin vitroassembly of CA tubes. Transmission electron microscopy and cryo-electron tomography demonstrated that the P122A and I124A mutations induce severe structural defects in the immature Gag lattice and abrogate conical core formation. Propagation of the P122A and I124A mutants in T-cell lines led to the selection of compensatory mutations within CA. Our findings demonstrate that the CA PPIP(122–125) loop comprises a structural element critical for the formation of the immature Gag lattice.IMPORTANCECapsid (CA) plays multiple roles in the HIV-1 replication cycle. CA-CA domain interactions are responsible for multimerization of the Gag polyprotein at virus assembly sites, and in the mature virion, CA monomers assemble into a conical core that encapsidates the viral RNA genome. Multiple CA regions that contribute to the assembly and release of HIV-1 particles have been mapped and investigated. Here, we identified and characterized a Pro-rich loop in CA that is important for the formation of the immature Gag lattice. Changes in this region disrupt viral production and abrogate the formation of infectious, mature virions. Propagation of the mutants in culture led to the selection of second-site compensatory mutations within CA. These results expand our knowledge of the assembly and maturation steps in the viral replication cycle and may be relevant for development of antiviral drugs targeting CA.

scholarly journals Relative contribution of non-structural protein 1 in dengue pathogenesis

2020 ◽  
Author(s):  
Pei Xuan Lee ◽  
Donald Heng Rong Ting ◽  
Clement Peng Hee Boey ◽  
Eunice Tze Xin Tan ◽  
Janice Zuo Hui Chia ◽  
...  
Keyword(s):  
Critical Role ◽  
Health Concern ◽  
Structural Protein ◽  
Promising Candidate ◽  
Relative Contribution ◽  
Structural Region ◽  
Strain Dependent ◽  
Pro Inflammatory Cytokines

AbstractDengue is a major public health concern in the tropical and sub-tropical world with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of sub-unit vaccine approaches, and the non-structural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non mouse-adapted DENV2 strains from the Cosmopolitan genotype that currently circulates in South-East Asia. Furthermore, exogenous administration of purified NS1 did not worsen in vivo vascular leakage in sub-lethally infected mice, thereby supporting that NS1 does not play a critical role in the pathogenesis of these DENV2 strains. Virus chimerization approaches indicated that the prME structural region, but not NS1, plays a critical role in driving in vivo fitness and virulence of the virus, through induction of key pro-inflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain-dependent, which warrants re-evaluation of NS1 as a universal dengue vaccine candidate.

scholarly journals Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

2020 ◽  
Author(s):  
Sergej Franz ◽  
Thomas Zillinger ◽  
Fabian Pott ◽  
Christiane Schüler ◽  
Sandra Dapa ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A ◽  
Chikungunya Virus ◽  
Protein S ◽  
Human Cells ◽  
Structural Protein ◽  
Infected Cells ◽  
Mayaro Virus ◽  
The Impact

AbstractInterferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-type IFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

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