scholarly journals The Amino-Conserved Domain of Human Cytomegalovirus UL80a Proteins Is Required for Key Interactions during Early Stages of Capsid Formation and Virus Production

2006 ◽  
Vol 81 (2) ◽  
pp. 620-628 ◽  
Author(s):  
Amy N. Loveland ◽  
Nang L. Nguyen ◽  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Capsid Protein ◽  
Infectious Virus ◽  
Major Capsid Protein ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Virus Production ◽  
Scaffolding Protein ◽  
Capsid Formation ◽  
Conserved Domain ◽  
Protease Precursor

ABSTRACT Assembly of many spherical virus capsids is guided by an internal scaffolding protein or group of proteins that are often cleaved and eliminated in connection with maturation and incorporation of the genome. In cytomegalovirus there are at least two proteins that contribute to this scaffolding function; one is the maturational protease precursor (pUL80a), and the other is the assembly protein precursor (pUL80.5) encoded by a shorter genetic element within UL80a. Yeast GAL4 two-hybrid assays established that both proteins contain a carboxyl-conserved domain that is required for their interaction with the major capsid protein (pUL86) and an amino-conserved domain (ACD) that is required for their self-interaction and for their interaction with each other. In the work reported here, we demonstrate that when the ACD is deleted (δACD) or disrupted by a point mutation (L47A), the bacterially expressed mutant protein sediments as a monomer during rate-velocity centrifugation, whereas the wild-type protein sediments mainly as oligomers. We also show that the L47A mutation reduces the production of infectious virus by at least 90%, results in the formation of irregular nuclear capsids, gives rise to tube-like structures in the nucleus that resemble the capsid core in cross-section and contain UL80 proteins, slows nuclear translocation of the major capsid protein, and may slow cleavage by the maturational protease. We provide physical corroboration that mutating the ACD disrupts self-interaction of the UL80 proteins and biological support for the proposal that the ACD has a critical role in capsid assembly and production of infectious virus.

scholarly journals Nuclear Localization Sequences in Cytomegalovirus Capsid Assembly Proteins (UL80 Proteins) Are Required for Virus Production: Inactivating NLS1, NLS2, or Both Affects Replication to Strikingly Different Extents

2008 ◽  
Vol 82 (11) ◽  
pp. 5381-5389 ◽  
Author(s):  
Nang L. Nguyen ◽  
Amy N. Loveland ◽  
Wade Gibson
Keyword(s):  
Nuclear Localization ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Virus Production ◽  
Wild Type Virus ◽  
Capsid Assembly ◽  
Scaffolding Proteins ◽  
Hcmv Replication ◽  
Protease Precursor ◽  
Nuclear Localization Sequences

ABSTRACT Scaffolding proteins of spherical prokaryotic and eukaryotic viruses have critical roles in capsid assembly. The primary scaffolding components of cytomegalovirus, called the assembly protein precursor (pAP, pUL80.5) and the maturational protease precursor (pPR, pUL80a), contain two nuclear localization sequences (NLS1 and NLS2), at least one of which is required in coexpression experiments to translocate the major capsid protein (MCP, pUL85) into the nucleus. In the work reported here, we have mutated NLS1 and NLS2, individually or together, in human cytomegalovirus (HCMV, strain AD169) bacmid-derived viruses to test their effects on virus replication. Consistent with results from earlier transfection/coexpression experiments, both single-mutant bacmids gave rise to infectious virus but the double mutant did not. In comparisons with the wild-type virus, both mutants showed slower cell-to-cell spread; decreased yields of infectious virus (3-fold lower for NLS1− and 140-fold lower for NLS2−); reduced efficiency of pAP, pPR, and MCP nuclear translocation (sixfold lower for NLS1− and eightfold lower for NLS2−); increased amounts of a 120-kDa MCP fragment; and reduced numbers of intranuclear capsids. All effects were more severe for the NLS2− mutant than the NLS1− mutant, and a distinguishing feature of cells infected with the NLS2− mutant was the accumulation of large, UL80 protein-containing structures within the nucleus. We conclude that these NLS assist in the nuclear translocation of MCP during HCMV replication and that NLS2, which is unique to the betaherpesvirus UL80 homologs, may have additional involvements during replication.

scholarly journals Assembly of the Herpes Simplex Virus Procapsid from Purified Components and Identification of Small Complexes Containing the Major Capsid and Scaffolding Proteins

1999 ◽  
Vol 73 (5) ◽  
pp. 4239-4250 ◽  
Author(s):  
William W. Newcomb ◽  
Fred L. Homa ◽  
Darrell R. Thomsen ◽  
Benes L. Trus ◽  
Naiqian Cheng ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Capsid Protein ◽  
Major Capsid Protein ◽  
Scaffolding Protein ◽  
Oligomeric State ◽  
Cell Extracts ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ∼30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

scholarly journals Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

2003 ◽  
Vol 77 (12) ◽  
pp. 7078-7092 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Subrata Barman ◽  
Tae Yong Yang ◽  
Debi P. Nayak
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Plaque Morphology ◽  
Wild Type ◽  
Wild Type Phenotype

ABSTRACT Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

scholarly journals Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

1995 ◽  
Vol 69 (10) ◽  
pp. 6567-6571 ◽  
Author(s):  
M Kawase ◽  
M Momoeda ◽  
N S Young ◽  
S Kajigaya
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Capsid Formation ◽  
B19 Parvovirus

scholarly journals Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

1998 ◽  
Vol 72 (10) ◽  
pp. 7722-7732 ◽  
Author(s):  
Scott M. Plafker ◽  
Wade Gibson
Keyword(s):  
Capsid Protein ◽  
Nuclear Localization ◽  
Major Capsid Protein ◽  
Nuclear Translocation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Protein Precursor ◽  
Nuclear Localization Signals ◽  
Carboxy Terminal

ABSTRACT The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499–509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in cisto translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in trans to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed.

scholarly journals Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

2003 ◽  
Vol 77 (5) ◽  
pp. 3247-3256 ◽  
Author(s):  
V. Brault ◽  
M. Bergdoll ◽  
J. Mutterer ◽  
V. Prasad ◽  
S. Pfeffer ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Aphid Transmission ◽  
Viral Rna ◽  
Beet Western Yellows Virus ◽  
Virus Accumulation ◽  
Capsid Formation ◽  
Arginine Residues ◽  
R Domain

ABSTRACT Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.

scholarly journals Identification of Chikungunya virus nucleocapsid core assembly modulators

2019 ◽  
Author(s):  
Sara E. Jones-Burrage ◽  
Zhenning Tan ◽  
Lichun Li ◽  
Adam Zlotnick ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Nucleic Acid ◽  
Capsid Protein ◽  
Chikungunya Virus ◽  
Infectious Virus ◽  
Virus Transmission ◽  
Virus Production ◽  
List Type ◽  
Dependent Manner ◽  
Particle Assembly

Abstract:The alphavirus Chikungunya virus is transmitted to humans via infected mosquitos. Most infected humans experience symptoms which can range from short-term fatigue and fever to debilitating arthritis that can last for months or years. Some patients relapse and experience symptoms months or years after the initial bout of disease. The capsid protein of Chikungunya virus forms a shell around the viral RNA genome; this structure is called the nucleocapsid core. The core protects the genome during virus transmission and with the correct environmental trigger, this proteinaceous shell dissociates and releases the viral genome to initiate infection. We hypothesized that targeting compounds to interfere with the nucleocapsid core’s function would constrain virus spread either by inhibiting the release of viral genomes during entry or by reducing the number of infectious virus particles assembled. We implemented a high throughput, in vitro, FRET-based assay to monitor nucleic acid packaging by purified Chikungunya capsid protein as a proxy for nucleocapsid core assembly and disassembly. We screened 10,000 compounds and found 45 that substantially modulated the assembly of core-like particles. A subset of compounds was selected to study their effects in virus-infected vertebrate cells. Our results show that four compounds inhibit infectious virus production by at least 90% in a dose-dependent manner. The most promising inhibitor was tested and found to reduce the amount of nucleocapsid cores inside the cell during Chikungunya virus infection. These compounds could be the foundation for anti-viral therapeutics.HighlightsA FRET-based assay to detect nucleic acid packaging by Chikungunya virus capsid proteinIdentification of small molecules that modulate core-like particle assemblyA subset of compounds that interfere with in vitro assembly also inhibit Chikungunya virus production in cell cultureIdentification of antiviral molecules that may not be identified by assays using reporter virusesPotential starting compounds for developing direct-acting antivirals

scholarly journals Nuclear Localization but Not PML Protein Is Required for Incorporation of the Papillomavirus Minor Capsid Protein L2 into Virus-Like Particles

2004 ◽  
Vol 78 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Katrin A. Becker ◽  
Luise Florin ◽  
Cornelia Sapp ◽  
Gerd G. Maul ◽  
Martin Sapp
Keyword(s):  
Capsid Protein ◽  
Nuclear Protein ◽  
Major Capsid Protein ◽  
Nuclear Translocation ◽  
Wild Type ◽  
Minor Capsid Protein ◽  
Virus Like Particles ◽  
Nuclear Domain ◽  
L1 Protein

ABSTRACT Recent reports suggest that nuclear domain(s) 10 (ND10) is the site of papillomavirus morphogenesis. The viral genome replicates in or close to ND10. In addition, the minor capsid protein, L2, accumulates in these subnuclear structures and recruits the major capsid protein, L1. We have now used cell lines deficient for promyelocytic leukemia (PML) protein, the main structural component of ND10, to study the role of this nuclear protein for L2 incorporation into virus-like particles (VLPs). L2 expressed in PML protein knockout (PML−/−) cells accumulated in nuclear dots, which resemble L2 aggregates forming at ND10 in PML protein-containing cells. These L2 assemblies also attracted L1 and the transcriptional repressor Daxx, suggesting that they are functional in the absence of PML protein. In addition, L2-containing VLPs assembled in PML−/− cells. In order to analyze whether incorporation of L2 into VLPs requires any specific subcellular localization, an L1 mutant defective for nuclear transport and L2 mutants deficient in nuclear translocation and/or ND10 localization were constructed. Using this approach, we identified two independent L2 domains interacting with L1. Mutant L2 proteins not accumulating in ND10 were incorporated into VLPs. Mutant L1 protein, which assembled into VLPs in the cytoplasm, did not incorporate L2 defective for nuclear translocation. The same mutant L2 protein, which passively diffuses into the nucleus, is incorporated into wild-type L1-VLPs in the nucleus. Our data demonstrate that the incorporation of L2 into VLPs requires nuclear but not ND10 localization.

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