Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for “humanized” stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.     

Utilization of 5-Bromouracil by Thymineless Bacteria

Several thymineless Escherichia coli strains have been examined for their ability to replicate their deoxyribonucleic acid when bromouracil is substituted for thymine. The procedure we describe was used to identify a thymineless strain with characteristics relatively favorable to its use in bromouracil labeling experiments. In addition, mutants with an “absolute” thymine requirement could be easily distinguished from one with a “leaky” thymine requirement.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

Mechanism of HIV-1 Virion Entrapment by Tetherin

Tetherin, an interferon-inducible membrane protein, inhibits the release of nascent enveloped viral particles from the surface of infected cells. However, the mechanisms underlying virion retention have not yet been fully delineated. Here, we employ biochemical assays and engineered tetherin proteins to demonstrate conclusively that virion tethers are composed of the tetherin protein itself, and to elucidate the configuration and topology that tetherin adopts during virion entrapment. We demonstrate that tetherin dimers adopt an “axial” configuration, in which pairs of transmembrane domains or pairs of glycosylphosphatidyl inositol anchors are inserted into assembling virion particles, while the remaining pair of membrane anchors remains embedded in the infected cell membrane. We use quantitative western blotting to determine that a few dozen tetherin dimers are used to tether each virion particle, and that there is ∼3- to 5-fold preference for the insertion of glycosylphosphatidyl inositol anchors rather than transmembrane domains into tethered virions. Cumulatively, these results demonstrate that axially configured tetherin homodimers are directly responsible for trapping virions at the cell surface. We suggest that insertion of glycosylphosphatidyl inositol anchors may be preferred so that effector functions that require exposure of the tetherin N-terminus to the cytoplasm of infected cells are retained.     

Composition and Function of Thylakoid Membranes from Grana-rich and Grana-deficient Chloroplast Mutants of Barley

Chlorophyll-deficient barley (Hordeum vulgare) mutants were studied that had chlorophyll a/b ratios either higher or lower than the wild type. Mutants with high ratios (>5.2) had a reduced proportion of their photosynthetic lamellae appressed into grana (“grana-deficient” mutants) compared with wild type (chlorophyll a/b = 3.2), while the majority of lamellae in the chloroplasts with low chlorophyll a/b ratios (2.0-2.4) were organized into grana (“grana-rich” mutants).     

The Emerging Role of Nuclear Viral DNA Sensors

Detecting pathogenic DNA by intracellular receptors termed “sensors” is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses.     

Mast Cells Expedite Control of Pulmonary Murine Cytomegalovirus Infection by Enhancing the Recruitment of Protective CD8 T Cells to the Lungs

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient Kit^W-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.     

Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity

Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.     

Comparative Phylogenomics and Evolution of the Brucellae Reveal a Path to Virulence

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as “stealth pathogens” that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing “atypical” strains and a highly conserved “classical” core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.     

Lower HIV Provirus Levels Are Associated with More APOBEC3G Protein in Blood Resting Memory CD4+ T Lymphocytes of Controllers In Vivo

Immunodeficiency does not progress for prolonged periods in some HLA B57- and/or B27-positive subjects with human immunodeficiency virus type 1 (HIV) infection, even in the absence of antiretroviral therapy (ART). These “controllers” have fewer HIV provirus-containing peripheral blood mononuclear cells than “non-controller” subjects, but lymphocytes that harbor latent proviruses were not specifically examined in studies to date. Provirus levels in resting memory cells that can serve as latent reservoirs of HIV in blood were compared here between controllers and ART-suppressed non-controllers. APOBEC3G (A3G), a cellular factor that blocks provirus formation at multiple steps if not antagonized by HIV virion infectivity factor (Vif), was also studied. HLA-linked HIV control was associated with less provirus and more A3G protein in resting CD4+ T central memory (Tcm) and effector memory (Tem) lymphocytes (provirus: p = 0.01 for Tcm and p = 0.02 for Tem; A3G: p = 0.02 for Tcm and p = 0.02 for Tem). Resting memory T cells with the highest A3G protein levels (>0.5 RLU per unit of actin) had the lowest levels of provirus (<1,000 copies of DNA per million cells) in vivo (p = 0.03, Fisher''s exact test). Using two different experimental approaches, Vif-positive viruses with more A3G were found to have decreased virion infectivity ex vivo. These results raise the hypothesis that HIV control is associated with increased cellular A3G that may be packaged into Vif-positive virions to add that mode of inhibition of provirus formation to previously described adaptive immune mechanisms for HIV control.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Membrane Uncoating of Intact Enveloped Viruses

Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.Enveloped virus entry at the plasma membrane includes binding of the virion to one or more receptors, changes in the virion components, membrane fusion, and membrane uncoating. The term “membrane uncoating” is being used to describe the separation of internal virion components from the viral membrane so the internal components can enter the cell. The term “uncoating” is sometimes used to mean the release of the viral genome from the capsid or other structures that have also entered the cell, but in this review, the term “membrane uncoating” will be used to represent only the separation of the virion internal contents and the viral envelope.Much of the original model of membrane fusion and uncoating was generally accepted as a result of a 1968 paper by Morgan and Howe (41). That paper provided strong evidence that Sendai virus (a paramyxovirus) entered a cell by fusion of the viral membrane with the cell plasma membrane. After membrane fusion, the virion rapidly lost its structure as the viral membrane merged with the host membrane and its components became part of the host membrane. The viral ribonucleoprotein (RNP) and internal proteins were released into the cytoplasm. This model of membrane uncoating is still generally accepted. For instance, in a 2007 virology text (24), this model was presented and illustrated with a figure from the Morgan and Howe paper. (The same figure is shown here as Fig. 2B.)Later, it was shown that Sendai viruses, which had been grown in fertilized chicken eggs, had different properties depending whether they had been harvested after growth for roughly 1 day (“early harvest”) or for several days (“late harvest”). The early-harvest viruses appear to be intact, but the late-harvest viruses have a different morphology and appear to be damaged (20, 26).This review summarizes data showing that intact early-harvest Sendai viruses uncoat quite differently from the way damaged late-harvest Sendai viruses uncoat. A model of intact paramyxovirus membrane uncoating is presented. The membrane uncoating of some other enveloped viruses that enter at the plasma membrane is compared to that described by this model.     

Induction of Humoral and Cell-Mediated Immune Responses by Hepatitis B Virus Epitope Displayed on the Virus-Like Particles of Prawn Nodavirus

Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide. Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the “a” determinant within HBsAg was displayed on the recombinant capsid protein of Macrobrachium rosenbergii nodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the “a” determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV “a” determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.     

Growth rate fluctuations in phycomyces sporangiophores

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input (“night-blind”) or output (“stiff” and “hypertropic”) of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.     

Using Multivalent Adenoviral Vectors for HIV Vaccination

Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the “antigen capsid-incorporation” strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His₆ within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His₆ epitope-specific humoral immune response.