ABSTRACT
The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of such global HIV-1 vaccine antigens has not previously been evaluated. Here, we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of infection following heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection correlated with vaccine-elicited binding, neutralizing, and functional nonneutralizing antibodies, suggesting that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy for the development of a global HIV-1 vaccine. PAPERCLIP:
Subject(s)
AIDS Vaccines/immunology , HIV-1 , Animals , Antibody Formation , Female , HIV Antigens/immunology , Human Immunodeficiency Virus Proteins/immunology , Immunity, Cellular , Macaca mulatta , Male , Molecular Sequence Data , Specific Pathogen-Free OrganismsABSTRACT
Human mpox (monkeypox), a disease with similarities to smallpox, is endemic in Africa where it has persisted as a zoonosis with limited human-to-human spread. Unexpectedly, the disease expanded globally in 2022 driven by human-to-human transmission outside of Africa. It is not yet known whether the latter is due solely to behavioral and environmental factors or whether the mpox virus is adapting to a new host. Genome sequencing has revealed differences between the current outbreak strains, classified as clade IIb, and the prior clade IIa and clade I viruses, but whether these differences contribute to virulence or transmission has not been determined. We demonstrate that the wild-derived inbred castaneous mouse provides an exceptional animal model for investigating clade differences in mpox virus virulence and show that the order is clade I > clade IIa > clade IIb.1. The greatly reduced replication of the clade IIb.1 major outbreak strain in mice and absence of lethality at 100 times the lethal dose of a closely related clade IIa virus, despite similar multiplication in cell culture, suggest that clade IIb is evolving diminished virulence or adapting to other species.
Subject(s)
Monkeypox virus , Mpox (monkeypox) , Humans , Mice , Animals , Monkeypox virus/genetics , Mpox (monkeypox)/epidemiology , Virulence/genetics , Models, Animal , Disease OutbreaksABSTRACT
Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.
Subject(s)
Poxviridae , Smallpox , Vaccinia , Animals , Cattle , Humans , Vaccinia virus/genetics , Serine Proteinase Inhibitors , Viral Proteins/genetics , DNA Replication , Host Specificity , DNA, Viral , Virus Replication , Receptors, VirusABSTRACT
Current vaccines have greatly diminished the severity of the COVID-19 pandemic, even though they do not entirely prevent infection and transmission, likely due to insufficient immunity in the upper respiratory tract. Here, we compare intramuscular and intranasal administration of a live, replication-deficient modified vaccinia virus Ankara (MVA)-based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) vaccine to raise protective immune responses in the K18-hACE2 mouse model. Using a recombinant MVA expressing firefly luciferase for tracking, live imaging revealed luminescence of the respiratory tract of mice within 6 h and persisting for 3 d following intranasal inoculation, whereas luminescence remained at the site of intramuscular vaccination. Intramuscular vaccination induced S-binding-Immunoglobulin G (IgG) and neutralizing antibodies in the lungs, whereas intranasal vaccination also induced Immunoglobulin A (IgA) and higher levels of antigen-specific CD3+CD8+IFN-γ+ T cells. Similarly, IgG and neutralizing antibodies were present in the blood of mice immunized intranasally and intramuscularly, but IgA was detected only after intranasal inoculation. Intranasal boosting increased IgA after intranasal or intramuscular priming. While intramuscular vaccination prevented morbidity and cleared SARS-CoV-2 from the respiratory tract within several days after challenge, intranasal vaccination was more effective as neither infectious virus nor viral messenger (m)RNAs were detected in the nasal turbinates or lungs as early as 2 d after challenge, indicating prevention or rapid elimination of SARS-CoV-2 infection. Additionally, we determined that neutralizing antibody persisted for more than 6 mo and that serum induced to the Wuhan S protein neutralized pseudoviruses expressing the S proteins of variants, although with less potency, particularly for Beta and Omicron.
Subject(s)
COVID-19 Vaccines , COVID-19 , Immunoglobulin A , Respiratory System , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vaccinia virus , Administration, Intranasal , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/prevention & control , COVID-19/transmission , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Mice , Mice, Transgenic , Respiratory System/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination/methods , Vaccinia virus/genetics , Vaccinia virus/immunologyABSTRACT
We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.
Subject(s)
Vaccinia virus , Vaccinia , Animals , Humans , Alkynes , Myristic Acid/metabolism , Vaccinia/metabolism , Vaccinia virus/genetics , Viral Proteins/metabolism , Virion/metabolism , Virus InternalizationABSTRACT
Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Genetic Vectors/genetics , SARS-CoV-2/immunology , Vaccines, DNA/immunology , Vaccinia virus/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Specificity/immunology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Disease Models, Animal , Gene Expression , Humans , Immunization , Immunization, Passive , Immunoglobulin G/immunology , Mice , Mice, Transgenic , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Vaccines, DNA/administration & dosage , Vaccines, DNA/geneticsABSTRACT
Modified vaccinia virus Ankara (MVA), a widely used vaccine vector for expression of genes of unrelated pathogens, is safe, immunogenic, and can incorporate large amounts of added DNA. MVA was derived by extensively passaging the chorioallantois vaccinia virus Ankara (CVA) vaccine strain in chicken embryo fibroblasts during which numerous mutations and deletions occurred with loss of replicative ability in most mammalian cells. Restoration of the deleted C12L gene, encoding serine protease inhibitor 1, enhances replication of MVA in human MRC-5 cells but only slightly in other human cells. Here we show that repair of the inactivated C16L/B22R gene of MVA enhances replication in numerous human cell lines. This previously uncharacterized gene is present at both ends of the genome of many orthopoxviruses and is highly conserved in most, including smallpox and monkeypox viruses. The C16L/B22R gene is expressed early in infection from the second methionine of the previously annotated Copenhagen strain open reading frame (ORF) as a 17.4-kDa protein. The C16/B22 and C12 proteins together promote MVA replication in human cells to levels that are comparable to titers in chicken embryo fibroblasts. Both proteins enhance virion assembly, but C16/B22 increases proteolytic processing of core proteins in A549 cells consistent with higher infectious virus titers. Furthermore, human A549 cells expressing codon-optimized C16L/B22R and C12L genes support higher levels of MVA replication than cell lines expressing neither or either alone. Identification of the genes responsible for the host-range defect of MVA may allow more rational engineering of vaccines for efficacy, safety, and propagation.
Subject(s)
Host Specificity , Vaccinia virus/physiology , Vaccinia/virology , Virus Replication , Amino Acid Sequence , Animals , Cell Line , Chick Embryo , Gene Deletion , Humans , Sequence Alignment , Vaccinia virus/genetics , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP ß-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.
Subject(s)
Poxviridae/physiology , Viral Fusion Proteins/metabolism , Virus Internalization , Animals , Cell Line , Cytoplasm/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Protein Binding , Vaccinia virus/genetics , Vaccinia virus/metabolism , Vaccinia virus/physiology , Viral Fusion Proteins/geneticsABSTRACT
Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.
Subject(s)
Nucleotidases/genetics , Nucleotidases/metabolism , Vaccinia virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Animals , Catalytic Domain , Cell Line , Chick Embryo , Chlorocebus aethiops , Homologous Recombination , Host Specificity , Humans , Nucleotidases/chemistry , Open Reading Frames , Point Mutation , RNA, Messenger/metabolism , RNA, Viral/metabolism , Sequence Deletion , Vaccinia virus/genetics , Viral Plaque Assay , Viral Proteins/chemistry , Virus ReplicationABSTRACT
Eleven highly conserved proteins comprise the poxvirus entry-fusion complex (EFC). We focused on vaccinia virus (VACV) O3, a 35-amino acid, largely hydrophobic component of unknown specific function. Experimental evolution was carried out by blindly passaging a virus that was severely impaired in entry due to deletion of the gene encoding O3. Large plaque variants that arose spontaneously were discerned by round four and their numbers increased thereafter. Genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. There were frame-shift mutations in consecutive Ts in open reading frames F9L and D8L and a nonsynonymous base substitution in L5R. F9 and L5 are EFC proteins and D8 is involved in VACV cell attachment. The F9L mutation occurred by round four in each of three independant passages, whereas the L5R and D8L mutations were detected only after nearly all of the genomes already had the F9L mutation. Viruses with deletions of O3L and single or double F9L, L5R and D8L mutations were constructed by homologous recombination. In a single round of infection, viruses with adaptive mutations including F9L alone or in combination exhibited statistically significant higher virus titers than the parental O3L deletion mutant or the L5R or D8L mutants, consistent with the order of selection during the passages. Further analyses indicated that the adaptive F9L mutants also had higher infectivities, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3.IMPORTANCE Entry into cells is an essential first step in virus replication and an important target of vaccine- elicited immunity. For enveloped viruses, this step involves the fusion of viral and host membranes to form a pore allowing entry of the genome and associated proteins. Poxviruses are unique in that this function is mediated by an entry-fusion complex (EFC) of eleven transmembrane proteins rather than by one or a few. The large number of proteins has hindered investigation of their individual roles. We focused on O3, a predominantly hydrophobic 35 amino acid component of the vaccinia virus EFC, and found that spontaneous mutations in the transmembrane domains of certain other entry proteins can partially compensate for the absence of O3. The mutants exhibited increased infectivity, entry and assembly or stability of the EFC.
ABSTRACT
The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.
Subject(s)
Interleukin-15/pharmacology , Killer Cells, Natural/immunology , Orthopoxvirus/immunology , Poxviridae Infections/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cytokines/immunology , Female , Immunity, Innate/drug effects , Interferon-gamma/immunology , Interleukin-15/immunology , Killer Cells, Natural/drug effects , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Orthopoxvirus/drug effects , Orthopoxvirus/pathogenicity , Poxviridae Infections/drug therapy , Signal Transduction/drug effects , Spleen/drug effects , Spleen/pathology , Spleen/virology , Vaccinia virus/immunologyABSTRACT
Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.
Subject(s)
RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Vaccinia virus/physiology , Virus Replication/physiology , A549 Cells , Animals , Chickens , Cytoplasm/metabolism , Cytoplasm/virology , DNA, Viral/genetics , DNA, Viral/metabolism , Gene Knockdown Techniques , Humans , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Seq , Repressor Proteins/geneticsABSTRACT
The entry/fusion complex (EFC) consists of 11 conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here, we describe serially passaging VACV in nonpermissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection, indicating the occurrence and enrichment of adaptive mutations. Sequencing of genomes of passaged and cloned viruses revealed mutations near the N terminus of the G9 open reading frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His-to-Arg mutation at amino acid 44 or a duplication of amino acids 26 to 39. An adaptive Tyr-to-Cys substitution at amino acid 42 was discovered using error-prone PCR to generate additional mutations. Myristoylation of G9 was unaffected by the near-N-terminal mutations. The roles of the G9 mutations in enhancing plaque size were validated by homologous recombination. The mutants exhibited enhanced entry and spread in A56/K2 cells and induced syncytia at neutral pH in HeLa cells despite the expression of A56/K2. The data suggest that the mutations perturb the interaction of G9 with A56/K2, although some association was still detected in detergent-treated infected cell lysates.IMPORTANCE The entry of enveloped viruses is achieved by the fusion of viral and cellular membranes, a critical step in infection that determines host range and provides targets for vaccines and therapeutics. Poxviruses encode an exceptionally large number of proteins comprising the entry/fusion complex (EFC), which enables infection of diverse cells. Vaccinia virus (VACV), the prototype member of the poxvirus family, also encodes the fusion regulatory proteins A56 and K2, which are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are nonpermissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2.
Subject(s)
Giant Cells/metabolism , Membrane Fusion/physiology , Mutation , Vaccinia virus/genetics , Vaccinia virus/physiology , Viral Proteins/genetics , Virus Internalization , Cell Line , Cell Membrane/metabolism , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions/physiology , Humans , Membrane Fusion/genetics , Poxviridae/genetics , Protein Interaction Domains and Motifs , Sequence Alignment , Vaccinia/metabolism , Vaccinia/virology , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolismABSTRACT
On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membraneless sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation, and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membraneless protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, relocalization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N terminus of the ATI or large truncations are made at the N or C termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation.IMPORTANCE Poxvirus genome replication, transcription, translation, and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.
Subject(s)
Vaccinia virus/metabolism , Viral Proteins/metabolism , Virus Replication/genetics , Cytoplasm/metabolism , DNA Replication , Eukaryotic Initiation Factor-4E/metabolism , HeLa Cells , Humans , Inclusion Bodies/metabolism , Inclusion Bodies, Viral/virology , Poxviridae/genetics , Poxviridae/metabolism , RNA Recognition Motif Proteins/genetics , RNA, Messenger/genetics , RNA-Binding Motifs/genetics , Ribosomes/metabolism , Vaccinia virus/genetics , Viral Proteins/genetics , Virion/metabolism , Virus Assembly/geneticsABSTRACT
Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios.IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.
Subject(s)
Adaptation, Biological/genetics , Frameshift Mutation , Myxoma virus/genetics , Transcription Factors/genetics , Vaccinia virus/genetics , Viral Proteins/genetics , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Codon, Nonsense , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Dosage , Genetic Fitness , Myxoma virus/metabolism , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombination, Genetic , Transcription Factors/metabolism , Vaccinia virus/metabolism , Viral Proteins/metabolism , Virus Replication , Whole Genome SequencingABSTRACT
Given the complex biology of human immunodeficiency virus (HIV) and its remarkable capacity to evade host immune responses, HIV vaccine efficacy may benefit from the induction of both humoral and cellular immune responses of maximal breadth, potency, and longevity. Guided by this rationale, we set out to develop an immunization protocol aimed at maximizing the induction of anti-Envelope (anti-Env) antibodies and CD8+ T cells targeting non-Env epitopes in rhesus macaques (RMs). Our approach was to deliver the entire simian immunodeficiency virus (SIV) proteome by serial vaccinations. To that end, 12 RMs were vaccinated over 81 weeks with DNA, modified vaccinia Ankara (MVA), vesicular stomatitis virus (VSV), adenovirus type 5 (Ad5), rhesus monkey rhadinovirus (RRV), and DNA again. Both the RRV and the final DNA boosters delivered a near-full-length SIVmac239 genome capable of assembling noninfectious SIV particles and inducing T-cell responses against all nine SIV proteins. Compared to previous SIV vaccine trials, the present DNA-MVA-VSV-Ad5-RRV-DNA regimen resulted in comparable levels of Env-binding antibodies and SIV-specific CD8+ T-cells. Interestingly, one vaccinee developed low titers of neutralizing antibodies (NAbs) against SIVmac239, a tier 3 virus. Following repeated intrarectal marginal-dose challenges with SIVmac239, vaccinees were not protected from SIV acquisition but manifested partial control of viremia. Strikingly, the animal with the low-titer vaccine-induced anti-SIVmac239 NAb response acquired infection after the first SIVmac239 exposure. Collectively, these results highlight the difficulties in eliciting protective immunity against immunodeficiency virus infection.IMPORTANCE Our results are relevant to HIV vaccine development efforts because they suggest that increasing the number of booster immunizations or delivering additional viral antigens may not necessarily improve vaccine efficacy against immunodeficiency virus infection.
Subject(s)
Immunity , Proteome , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/immunology , Animals , Antibodies, Neutralizing , Antibodies, Viral/immunology , Antigens, Viral , CD8-Positive T-Lymphocytes/immunology , Humans , Immunization, Secondary , Macaca mulatta/immunology , Vaccination , Viral Load , ViremiaABSTRACT
Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.
Subject(s)
Host Specificity/immunology , Serine Proteinase Inhibitors/immunology , Vaccinia virus/physiology , Vaccinia/virology , Viral Vaccines/immunology , Virus Replication , A549 Cells , Genetic Vectors/immunology , Humans , Serine Proteinase Inhibitors/genetics , Vaccinia/immunology , Vaccinia/prevention & controlABSTRACT
Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+ DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.
Subject(s)
Apoptosis/genetics , Gene Products, gag/genetics , Gene Products, pol/genetics , Vaccinia virus/genetics , Vaccinia virus/immunology , env Gene Products, Human Immunodeficiency Virus/genetics , Animals , Cell Line, Tumor , Dendritic Cells/immunology , Female , HeLa Cells , Humans , Macaca mulatta , Mice , Mice, Inbred BALB C , Viral Proteins/genetics , Viral Vaccines/immunology , env Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbitpox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI-1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data-replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)-were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells.
Subject(s)
Interferon Regulatory Factor-2/metabolism , Orthopoxvirus/physiology , Receptors, Virus/metabolism , Replication Protein C/metabolism , Serpins/genetics , A549 Cells , Humans , Microarray Analysis , Mutation , Orthopoxvirus/enzymology , Orthopoxvirus/genetics , Poxviridae Infections/metabolism , Poxviridae Infections/virology , Viral Proteins/genetics , Virus ReplicationABSTRACT
The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.