scholarly journals Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

2009 ◽  
Vol 83 (18) ◽  
pp. 9370-9387 ◽  
Author(s):  
Hyung Suk Oh ◽  
Harsh B. Pathak ◽  
Ian G. Goodfellow ◽  
Jamie J. Arnold ◽  
Craig E. Cameron
Keyword(s):  
Cleavage Site ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Ectopic Expression ◽  
Virus Production ◽  
Genome Replication ◽  
Multiplication Cycle ◽  
Final Yield ◽  
Reduced Rate ◽  
Insight Into

ABSTRACT A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

scholarly journals Mutations in the Yellow Fever Virus Nonstructural Protein NS2A Selectively Block Production of Infectious Particles

2002 ◽  
Vol 76 (10) ◽  
pp. 4773-4784 ◽  
Author(s):  
Beate M. Kümmerer ◽  
Charles M. Rice
Keyword(s):  
Amino Acids ◽  
Yellow Fever ◽  
Cleavage Site ◽  
Infectious Virus ◽  
Yellow Fever Virus ◽  
Nonstructural Protein ◽  
Virus Production ◽  
Fever Virus ◽  
Helicase Domain ◽  
Infected Cells

ABSTRACT Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aα, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK↓T within NS2A. Exchange of serine for lysine at this site (QKT→QST) blocks the production of both NS2Aα and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aα. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aα was replaced with serine. In addition to true reversions, NS2Aα cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aα cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aα site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles.

scholarly journals Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production

2006 ◽  
Vol 80 (16) ◽  
pp. 8178-8189 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Virus Particles ◽  
M1 Protein

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.

scholarly journals Roles of ESCRT proteins (ALIX and CHIMP4A) and their interplay with ISG15 during tick-borne flavivirus infection

2021 ◽  
Author(s):  
Pham-Tue-Hung Tran ◽  
Abhilash I. Chiramel ◽  
Magnus Johansson ◽  
Wessam Melik
Keyword(s):  
Virus Replication ◽  
Defense Mechanisms ◽  
Virus Assembly ◽  
Ectopic Expression ◽  
Virus Production ◽  
Host Cells ◽  
Diverse Range ◽  
Positive Effects ◽  
Ns3 Protein ◽  
Flavivirus Infection

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we show that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required for flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrate specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical methodology, we show that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT protein, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection the virus employs ESCRT protein members for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Other ESCRT protein members such as TSG101 are not required for virus production. The ESCRT, ALIX -CHMP4A complex, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

scholarly journals Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

2009 ◽  
Vol 84 (4) ◽  
pp. 1666-1673 ◽  
Author(s):  
Martina Kopp ◽  
Catherine L. Murray ◽  
Christopher T. Jones ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Virus Production ◽  
Terminal Region ◽  
Dual Processing ◽  
Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

scholarly journals Yellow Fever Virus NS3 Plays an Essential Role in Virus Assembly Independent of Its Known Enzymatic Functions

2008 ◽  
Vol 82 (7) ◽  
pp. 3342-3352 ◽  
Author(s):  
Chinmay G. Patkar ◽  
Richard J. Kuhn
Keyword(s):  
Yellow Fever ◽  
Infectious Virus ◽  
Yellow Fever Virus ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Fever Virus ◽  
Helicase Domain ◽  
Genome Replication ◽  
Wild Type ◽  
Nonstructural Proteins

ABSTRACT In flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. It was previously reported that mutations in yellow fever virus (YFV) nonstructural protein NS2A blocked production of infectious virus and that this block could be released by a suppressor mutation in NS3. Here, based on studies using a YFV replicon-based trans-packaging system as well as full-length YFV cDNA, we report that mutation of a conserved tryptophan at position 349 in the helicase domain of NS3 blocks production of infectious virus particles, revealing an as-yet-unknown role for NS3 in virus assembly. Mutation of tryptophan 349 to alanine (W349A) had no effect on viral replication, as demonstrated by wild-type levels of viral RNA amplification and protein expression in W349A-transfected cells. Although release of infectious virus was not detected, release of capsidless subviral particles was not blocked. The assembly defect in W349A could be trans-complemented inefficiently using BHK-REP cells (a cell line containing persistently replicating YFV replicon RNA). trans-complementation was also demonstrated by supplying wild-type NS2B-3 or NS3 protein alone as well as by supplying inactive NS2B-3 protein, indicating that this function of NS3 in virus assembly was independent of its known enzymatic functions.

scholarly journals Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1523
Author(s):  
Huining Pang ◽  
Ling Li ◽  
Hongru Liu ◽  
Zishu Pan
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Infectious Virus ◽  
Virus Production ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Replication ◽  
Rdrp Activity ◽  
Viral Genome Replication

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

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

2008 ◽  
Vol 82 (15) ◽  
pp. 7624-7639 ◽  
Author(s):  
Yinghong Ma ◽  
Jeremy Yates ◽  
Yuqiong Liang ◽  
Stanley M. Lemon ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplets ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Virus Production ◽  
Intermediate Step ◽  
Particle Assembly ◽  
Chimeric Rna

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.

scholarly journals Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Hsuan Liu ◽  
Michael L. Grantham ◽  
Andrew Pekosz
Keyword(s):  
Virus Particle ◽  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Filament Formation ◽  
Virus Budding ◽  
Virus Particles

ABSTRACTThe influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine-to-alanine mutation at residue 76 (Y76A), were essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation, 93/244) that are not found in circulating influenza virus strains or have not been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest that influenza A virus budding and genome incorporation can occur independently and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.IMPORTANCEInfluenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this work, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency with which virus particles bud from infected cells and restoring filament formation at the infected-cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all, aspects of virus particle assembly.

scholarly journals Differential replication of two porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression

2014 ◽  
Vol 95 (4) ◽  
pp. 910-921 ◽  
Author(s):  
Sandra Fernandes ◽  
Maude Boisvert ◽  
Jozsef Szelei ◽  
Peter Tijssen
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Infectious Virus ◽  
Virus Production ◽  
Productive Infection ◽  
Structural Proteins ◽  
Porcine Parvovirus ◽  
Genome Replication ◽  
Initial Generation ◽  
Cellular Components

Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5 kb genome expresses three major non-structural proteins (NS1, NS2 and SAT), and two structural proteins (VP1 and VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines have been shown to replicate the virus efficiently. Cell lines were established from a primary culture of bovine cells that allowed allotropic variants of PPV to be distinguished. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared with porcine cells, while production of infectious virus of the Kresse strain was barely detectable. Restriction points in these cells were the initial generation of DNA replication intermediates and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant were also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains, although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlight the dependent nature of parvovirus tropism on host factors and suggest that cells from a non-host origin can fully support a productive infection by both strains.

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