MinKyung Yi

The University of Texas Medical Branch at Galveston

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Stanley M. Lemon

University of North Carolina at Chapel Hill

Christoph Welsch

Goethe University Frankfurt

Tetsuro Shimakami

Kanazawa University Hospital

Saravanabalaji Shanmugam

The University of Texas Medical Branch at Galveston

Nigel Bourne

The University of Texas Medical Branch at Galveston

Daisuke Yamane

Tokyo Metropolitan Institute of Medical Science

Rohit K. Jangra

Louisiana State University Health Sciences Center Shreveport

Meital Gal-Tanamy

Bar-Ilan University

Itai Benhar

Tel Aviv University

Masao Honda

Kanazawa University Hospital

Cooperative Institutions

The University of Texas Medical Branch at Galveston

University of North Carolina at Chapel Hill

Kanazawa University

Goethe University Frankfurt

Kanazawa University Hospital

UNC Lineberger Comprehensive Cancer Center

Tel Aviv University

University Hospital Frankfurt

National Institutes of Health

Consorci Institut D'Investigacions Biomediques August Pi I Sunyer

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Hepatitis C Virus NS2 Protein Serves as a Scaffold for Virus Assembly by Interacting with both Structural and Nonstructural Proteins

Journal of Virology (2010)

Yinghong Ma Manu Anantpadma Jennifer M. Timpe Saravanabalaji Shanmugam Sher M. Singh

Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets, where virus particle assembly is thought to occur. p7 may play an accessory role by regulating NS2 membrane topology, which is important for NS2-mediated protein interactions and therefore NS2 function.

NS3

NS5A

10.1128/jvi.01070-10

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Citations (157)

Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Journal of Virology (2013)

Saravanabalaji Shanmugam MinKyung Yi

Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD.

NS3

NS2-3 protease

Signal peptidase

10.1128/jvi.01807-13

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Citations (18)

345 INHIBITION OF HCV CELL CULTURE INFECTIOUS VIRUS AND DRUG RESISTANT REPLICONS BY INTRABODIES

Journal of Hepatology (2009)

Meital Gal-Tanamy Romy Zemel L. Bachmetov Rohit K. Jangra Rodrigo A. Villanueva

Replicon

10.1016/s0168-8278(09)60347-0

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In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

Proceedings of the National Academy of Sciences (2008)

Meital Gal-Tanamy Zhen‐Yong Keck MinKyung Yi Jane A. McKeating Arvind H. Patel

Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.

Viral quasispecies

Viral evolution

10.1073/pnas.0809879105

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Citations (86)

Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function

Journal of Virology (2000)

MinKyung Yi Derk E. Schultz Stanley M. Lemon

ABSTRACT Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involved in glycolysis, binds specifically to several viral RNAs, but the functional significance of this interaction is uncertain. Both GAPDH and polypyrimidine tract binding protein (PTB) bind to overlapping sites in stem-loop IIIa of the internal ribosome entry site (IRES) of Hepatitis A virus (HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary structure, we reasoned that GAPDH may suppress the ability of the IRES to direct cap-independent translation, making its effects antagonistic to the translation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. Lemon, J. Biol. Chem. 271:14134–14142, 1996). To test this hypothesis, we constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and a downstream Renilla luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. RLuc activity was compared with that from a control, null-expression plasmid that was identical except for a frameshift mutation within the 5′ GAPDH coding sequence. In transfection experiments, GAPDH overexpression significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells but not in Huh-7 cells, which have a significantly greater cytoplasmic abundance of PTB. GAPDH suppression of HAV translation was greater with the wild-type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P16) that has reproducibly higher basal translational activity in BSC-1 cells. Stem-loop IIIa RNA from the latter IRES had significantly lower affinity for GAPDH in filter binding experiments. Thus, the binding of GAPDH to the IRES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells. The enhanced replication capacity of cell culture-adapted HAV in such cells may be due in part to reduced affinity of the viral IRES for GAPDH.

Glyceraldehyde 3-phosphate dehydrogenase

Polypyrimidine tract-binding protein

Picornavirus

10.1128/jvi.74.14.6459-6468.2000

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Citations (98)

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system

Antiviral Research (2005)

Nigel Bourne Richard B. Pyles MinKyung Yi Ronald L. Veselenak Melissa Davis

Replicon

Subgenomic mRNA

10.1016/j.antiviral.2005.03.006

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Citations (35)

Structure–function analysis of the 3′ stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

RNA (2003)

MinKyung Yi Stanley M. Lemon

Previous studies indicate that the 3′ terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3′ terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the −19 and −28 positions (from the 3′ end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3′ terminus. Further experiments indicated a strong preference for U at the 3′ terminal position (followed in order by C, A, and G), and a G at the −2 position. These features of stem-loop I are likely to facilitate recognition of the 3′ end of the viral RNA by the viral RNA replicase.

Stem-loop

Replication

Nucleic acid structure

10.1261/rna.2144203

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Citations (99)

miR-122 does not modulate the elongation phase of hepatitis C virus RNA synthesis in isolated replicase complexes

Antiviral Research (2010)

Rodrigo A. Villanueva Rohit K. Jangra MinKyung Yi R. B. Pyles Nigel Bourne

Replicon

NS5A

NS5B

RNA Silencing

10.1016/j.antiviral.2010.07.004

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Citations (47)

Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition

Journal of Virology (2003)

Wenyan Wang Frederick Lahser MinKyung Yi Jacquelyn Wright-Minogue Ellen Xia

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.

NS3

Replicon

NS5A

NS2-3 protease

10.1128/jvi.78.2.700-709.2004

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Citations (22)

NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly

Journal of Virology (2008)

Yinghong Ma Jeremy Yates Yuqiong Liang Stanley M. Lemon MinKyung Yi

ABSTRACT A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

NS3

NS5A

RNA Helicase A

NS2-3 protease

10.1128/jvi.00724-08

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Citations (195)