The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.     

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.     

Identification of basic amino acids at the N-terminal end of the core protein that are crucial for hepatitis C virus infectivity

A major function of the hepatitis C virus (HCV) core protein is the interaction with genomic RNA to form the nucleocapsid, an essential component of the virus particle. Analyses to identify basic amino acid residues of HCV core protein, important for capsid assembly, were initially performed with a cell-free system, which did not indicate the importance of these residues for HCV infectivity. The development of a cell culture system for HCV (HCVcc) allows a more precise analysis of these core protein amino acids during the HCV life cycle. In the present study, we used a mutational analysis in the context of the HCVcc system to determine the role of the basic amino acid residues of the core protein in HCV infectivity. We focused our analysis on basic residues located in two clusters (cluster 1, amino acids [aa]6 to 23; cluster 2, aa 39 to 62) within the N-terminal 62 amino acids of the HCV core protein. Our data indicate that basic residues of the first cluster have little impact on replication and are dispensable for infectivity. Furthermore, only four basic amino acids residues of the second cluster (R50, K51, R59, and R62) were essential for the production of infectious viral particles. Mutation of these residues did not interfere with core protein subcellular localization, core protein-RNA interaction, or core protein oligomerization. Moreover, these mutations had no effect on core protein envelopment by intracellular membranes. Together, these data indicate that R50, K51, R59, and R62 residues play a major role in the formation of infectious viral particles at a post-nucleocapsid assembly step.     

Two domains that control prefusion stability and transport competence of the measles virus fusion protein

Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.     

Mutations in the E1 hydrophobic domain of rubella virus impair virus infectivity but not virus assembly

Rubella virus (RV) virions contain three structural proteins, a capsid protein that interacts with viral genomic RNA to form a nucleocapsid and two membrane glycoproteins, E2 and E1. We found that substitution of either an aspartic acid residue at Gly93 (G93D) or a glycine residue at Pro104 (P104G) in the internal hydrophobic domain of E1 affected virus infectivity but not virus assembly. Viruses carrying G93D and P104G mutations had impaired infectivity, reduced 1,000-fold and 10-fold, respectively. A revertant was isolated from the G93D mutant. Sequencing analysis showed that the substituted aspartic acid residue in G93D mutant had reverted to the original glycine residue, suggesting the involvement of Gly93 in membrane fusion during viral entry.     

Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange

Hepatitis delta virus (HDV) particles are coated with the envelope proteins (large, middle, and small) of the hepatitis B virus (HBV). The large protein bears an infectivity determinant in its pre-S1 domain, whereas a second determinant has been proposed to map to the cysteine-rich antigenic loop (AGL) within the S domain of all three envelope proteins (G. Abou Jaoudé and C. Sureau, J. Virol. 79:10460-10466, 2006). In this study, the AGL cysteines were substituted by serine or alanine, and the mutants were evaluated for their function at viral entry using HDV particles and susceptible HepaRG cells. Mutations of cysteines 121 to 149 were tolerant of the production of HDV virions. The mutations altered the structure and antigenicity of the conserved “a” determinant of the AGL, as measured by conformation-sensitive antibodies, and they created a block to infectivity. Substitution of Cys-90 or Cys-221, located outside of the AGL, had no impact on the “a” determinant or viral entry. Furthermore, infectivity was maintained when the AGL CxxC motif at position 121 to 124 was modified by single-amino-acid deletion or insertion, suggesting that cysteines 121 and 124 are not catalyzers of thiol/disulfide exchange. However, membrane-impermeable inhibitors of thiol/disulfide isomerazation demonstrated a dose-dependent inhibition of infection in an in vitro assay when applied to the virus prior to inoculation or during the virus-cell interaction period. Overall, the results demonstrate the essential role of the AGL cysteines at viral entry, and they establish a correlation between the cysteine disulfide network, the conformation of the “a” determinant, and infectivity.     

Role of human immunodeficiency virus type 1 matrix phosphorylation in an early postentry step of virus replication

The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.     

Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus

Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the world's population. Recent advances have enabled in vitro propagation of the virus and allow assembly and egress to be investigated for the first time. As a component of the virion, the HCV core protein likely functions primarily in infectious virus production, although little is known about the determinants of this activity. To investigate the roles of core in the viral life cycle, we performed a comprehensive deletion and alanine scanning mutagenesis study of this protein in the context of a genotype 2a reporter virus. We have confirmed that core protein is essential for infectious virion production and have identified numerous residues required for this role. The infectivity of several assembly-defective core mutants could be rescued by compensatory mutations identified in p7 and NS2, suggesting genetic interactions with core and highlighting the importance of these nonstructural proteins in infectious virion morphogenesis.     

Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA "coat" can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.     

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.     

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.     

Full-length sequence analysis of subacute sclerosing panencephalitis (SSPE) virus, a mutant of measles virus, isolated from brain tissues of a patient shortly after onset of SSPE

Subacute sclerosing panencephalitis (SSPE) virus, a measles virus (MeV) mutant, was isolated from brain tissues of a patient shortly after the clinical onset, and the entire viral genome was sequenced. The virus, named SSPE-Kobe-1, formed syncytia on B95a and Vero/SLAM cells without producing cell-free infectious virus particles, which is characteristic of SSPE virus. Phylogenetic analysis classified SSPE-Kobe-1 into genotype D3. When compared with an MeV field isolate of the same genotype (Ich-B strain), SSPE-Kobe-1 exhibited mutation rates of 0.8-1.6% at the nucleotide level in each of the proteincoding regions of the viral genome. It is noteworthy that the mutation rate of the M gene (1.2%) of SSPE-Kobe-1 was considerably lower than for other SSPE virus strains reported so far, but that the majority of the mutations (75%) were the uridine-to-cytidine biased hypermutation characteristic of the SSPE virus M gene. At the amino acid level, the viral proteins, such as N, P, C, V, M, F, H and L proteins, had point-mutations on 3, 7, 1, 4, 3, 9, 8 and 14 residues, respectively, compared with the Ich-B strain. In addition, the F and H proteins had mutated C-termini due to single-point mutations near or at the stop codons. Two of the three mutations in the M protein were Leu-to-Pro mutations, which are likely to affect the conformation and, therefore, the function of the protein. Because of the relatively small number of mutations, SSPE-Kobe-1 would be a useful tool to study genetic evolution of SSPE virus.     

The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism

The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.     

Structural tolerance versus functional intolerance to mutation of hydrophobic core residues surrounding cavities in a parvovirus capsid

The structural and functional relevance of amino acid residues surrounding cavities within the hydrophobic core of the protein subunits that form the capsid of parvoviruses has been investigated. Several of the evolutionarily conserved, hydrophobic residues that delimit these cavities in the capsid of the minute virus of mice were replaced by other hydrophobic residues that would affect the size and/or shape of the cavity. When four or more methylene-sized groups were introduced, or six or more groups removed, capsid assembly was drastically impaired. In contrast, the introduction or removal of up to three groups had no significant effect on capsid assembly or thermostability. However, many of these mutations affected a capsid conformational transition needed for viral infectivity. Replacement of some polar residues around the largest cavity showed that capsid assembly requires a carboxylate buried within this cavity, but both aspartate and glutamate are structurally accepted. Again, only the aspartate allowed the production of infectious viruses, because of a specific role in encapsidation of the viral genome. These observations provide evidence of a remarkable structural tolerance to mutation of the hydrophobic core of the protein subunits in a viral capsid, and of an involvement of core residues and internal cavities in capsid functions needed for infectivity.     

A genetic interaction between the core and NS3 proteins of hepatitis C virus is essential for production of infectious virus

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1(T)-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1(T)-64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1(T)-64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

Generation of a cell culture-adapted hepatitis C virus with longer half life at physiological temperature

Background

We previously reported infectious HCV clones that contain the convenient reporters, green fluorescent protein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferation and screening of anti-HCV drugs, the infectivity and yield of the viruses were low.

Methodology/Principal Findings

In order to obtain a highly efficient HCV cultivation system, we transfected Huh7.5.1 cells [1] with JFH 5a-GFP RNA and then cultivated cells for 20 days. We found a highly infectious HCV clone containing two cell culture-adapted mutations. Two cell culture-adapted mutations which were responsible for the increased viral infectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ∼100-fold higher than that of the parental virus. The mutation in the E2 protein increased the viability of virus at 37°C by acquiring prolonged interaction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ∼2.5 to 3 hours at 37°C. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6 hours at 37°C. The mutation in the p7 protein, either singly or in combination with the E2 mutation, enhanced infectious virus production about 10–50-fold by facilitating an early step of virion production.

Conclusion/Significance

The mutation in the E2 protein generated by the culture system increases virion viability at 37°C. The adaptive mutation in the p7 protein facilitates an earlier stage of virus production, such as virus assembly and/or morphogenesis. These reporter-containing HCV viruses harboring adaptive mutations are useful in investigations of the viral life cycle and for developing anti-viral agents against HCV.     

Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.