Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

The major yolk proteins of higher Diptera are homologs of a class of minor yolk proteins in lepidoptera

In most oviparous animals, including insects, vitellogenin (Vg) is the major yolk protein precursor. However, in the higher Diptera (cyclorrhaphan flies), a class of proteins homologous to lipoprotein lipases called yolk polypeptides (YP) are accumulated by oocytes instead of Vg, which is not produced at all. Lepidopterans (moths) produce Vg as the major yolk protein precursor, but also manufacture a class of minor yolk proteins referred to as egg-specific proteins (ESP) or YP2s. Although the lepidopteran ESP/YP2s are related to lipoprotein lipases, previous attempts to directly demonstrate their homology with higher-dipteran YPs were unsuccessful. In this paper, a multiple alignment of amino acid sequences was constructed using a shared lipid binding motif as an anchor, to demonstrate that lepidopteran ESP/YP2s, higher-dipteran YPs, and lipoprotein lipases are indeed homologous. Phylogenetic analyses of the aligned sequences were performed using both distance-based and parsimony strategies. It is apparent that the higher dipterans did not requisition a lipoprotein lipase to replace Vg as a yolk protein precursor, but instead utilize a class of proteins with an evolutionary history of use as minor constituents of yolk in other insects.     

Covariation of major and minor viral capsid proteins in norovirus genogroup II genotype 4 strains

We report sequence hypervariability in the viral protein 1 (VP1) interaction domain of VP2 in the norovirus (NoV) genogroup II genotype 4 (GII.4) lineage on 3 levels: (i) the global evolution of pandemic/epidemic strains from the mid-1970s through post-2006, (ii) the local emergence of an epidemic strain, and (iii) an immunocompromised patient chronically shedding NoV. When a quantitative yeast two-hybrid assay was used, VP2 was found to interact with VP1 in a time-ordered, strain-dependent manner among 3 NoV GII.4 strains. Our findings suggest that VP1 and VP2 may covary in virus evolution and that sequence hypervariability of VP2 may be functionally driven. Further investigations are warranted.     

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell’s exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.     

NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture

Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.     

Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins

The elicitation of broadly and efficiently neutralizing antibodies in humans by active immunization is still a major obstacle in the development of vaccines against pathogens such as the human immunodeficiency virus (HIV), influenza virus, hepatitis C virus or cytomegalovirus. Here, we describe a mammalian cell surface display and monoclonal antibody (mAb)-mediated panning technology that allows affinity-based selection of envelope (Env) variants from libraries. To this end, we established an experimental setup featuring: 1) single and site specific integration of Env to link genotype and phenotype, 2) inducible Env expression to avoid cytotoxicity effects, 3) translational coupling of Env and enhanced green fluorescent protein expression to normalize for Env protein levels, and 4) display on HEK cells to ensure native folding and mammalian glycosylation. For proof of concept, we applied our method to a chimeric HIV-1 Env model library comprising variants with differential binding affinities to the V3-loop-directed mAbs 447–52D and HGN194. Fluorescence-activated cell sorting selectively enriched a high affinity variant up to 56- and 55-fold for 447–52D and HGN194, respectively, after only a single round of panning. Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. The binding profiles of membrane-bound gp145 and soluble gp140 chimeras showed identical affinity ranking, suggesting that the technology can guide the identification of Env variants with optimized antigenic properties for subsequent use as vaccine candidates. Finally, our mAb-based cellular display and selection strategy may also prove useful for the development of prophylactic vaccines against pathogens other than HIV.     

RabGEFs are a major determinant for specific Rab membrane targeting

Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.     

An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.

Host cell proteases responsible for activation of viral fusion glycoproteins are an important determinant for spread and tropism of various animal viruses. Exemplifying such proteases for the first time, we isolated an endoprotease from chick embryo, that activates para- and orthomyxovirus fusion glycoproteins by cleaving their precursor proteins at a specific, single arginine site. The protease is a calcium dependent serine protease consisting of two subunits, the 33 kd catalytic chain and the 23 kd chain possibly required for Ca2+ binding, and was found to be highly homologous, if not identical, to the blood clotting factor X(FX), a member of the prothrombin family. Its high efficiency and specificity in cleavage reactions was attributable to the properties characteristic of FX. Its role in vivo was strongly supported by cleavage inhibition in ovo highly selective for this virus group with a specific peptide inhibitor against FX.     

Alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development

Macrophages are abundant in the lower respiratory tract. They play a central role in the innate response to infection but may also modulate excessive inflammation. Both macrophages and ciliated epithelial cells respond to infection by releasing soluble mediators, leading to the recruitment of innate and adaptive effector cells. To study the role of lung macrophages in acute respiratory viral infection, we depleted them by the inhalation of clodronate liposomes in an established mouse model of respiratory syncytial virus (RSV) disease. Infection caused an immediate local release of inflammatory cytokines and chemokines, peaking on day 1, which was virtually abolished by clodronate liposome treatment. Macrophage depletion inhibited the activation (days 1 to 2) and recruitment (day 4) of natural killer (NK) cells and enhanced peak viral load in the lung (day 4). However, macrophage depletion did not affect the recruitment of activated CD4 or CD8 T cells, weight loss, or virus-induced changes in lung function. Therefore, lung macrophages play a central role in the early responses to viral infection but have remarkably little effect on the adaptive response occurring at the time of peak disease severity.     

Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity.

Disulfide bonds were found to link the nonglycosylated envelope protein VP-2/M (19 kDa), encoded by open reading frame 6, and the major envelope glycoprotein VP-3 (25 to 42 kDa), encoded by open reading frame 5, of lactate dehydrogenase-elevating virus (LDV). The two proteins comigrated in a complex of 45 to 55 kDa when the virion proteins were electrophoresed under nonreducing conditions but dissociated under reducing conditions. Furthermore, VP-2/M was quantitatively precipitated along with VP-3 in this complex by three neutralizing monoclonal antibodies to VP-3. The infectivity of LDV was rapidly and irreversibly lost during incubation with 5 to 10 mM dithiothreitol (> 99% in 6 h at room temperature), which is known to reduce disulfide bonds. LDV inactivation correlated with dissociation of VP-2/M and VP-3. The results suggest that disulfide bonds between VP-2/M and VP-3 are important for LDV infectivity. Hydrophobic moment analyses of the predicted proteins suggest that VP-2/M and VP-3 both possess three adjacent transmembrane segments and only very short ectodomains (10 and 32 amino acids, respectively) with one and two cysteines, respectively. Inactivation of LDV by dithiothreitol and dissociation of the two envelope proteins were not associated with alterations in LDV's density or sedimentation coefficient.     

Intralysosomal iron: a major determinant of oxidant-induced cell death

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH(4)Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH(4)Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H(2)O(2), and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH(4)Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H(2)O(2) addition, was absent in cells preexposed to 10 mM NH(4)Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.     

Envelope is a major viral determinant of the distinct in vitro cellular transformation tropism of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are related deltaretroviruses but are distinct in their disease-inducing capacity. These viruses can infect a variety of cell types, but only T lymphocytes become transformed, which is defined in vitro as showing indefinite interleukin-2-independent growth. Studies have indicated that HTLV-1 has a preferential tropism for CD4+ T cells in vivo and is associated with the development of leukemia and neurological disease. Conversely, the in vivo T-cell tropism of HTLV-2 is less clear, although it appears that CD8+ T cells preferentially harbor the provirus, with only a few cases of disease association. The difference in T-cell transformation tropism has been confirmed in vitro as shown by the preferential transformation of CD4+ T cells by HTLV-1 versus the transformation of CD8+ T cells by HTLV-2. Our previous studies showed that Tax and overlapping Rex do not confer the distinct T-cell transformation tropisms between HTLV-1 and HTLV-2. Therefore, for this study HTLV-1 and HTLV-2 recombinants were generated to assess the contribution of LTR and env sequences in T-cell transformation tropism. Both sets of proviral recombinants expressed p19 Gag following transfection into cells. Furthermore, recombinant viruses were replication competent and had the capacity to transform T lymphocytes. Our data showed that exchange of the env gene resulted in altered T-cell transformation tropism compared to wild-type virus, while exchange of long terminal repeat sequences had no significant effect. HTLV-2/Env1 preferentially transformed CD4+ T cells similarly to wild-type HTLV-1 (wtHTLV-1), whereas HTLV-1/Env2 had a transformation tropism similar to that of wtHTLV-2 (CD8+ T cells). These results indicate that env is a major viral determinant for HTLV T-cell transformation tropism in vitro and provides strong evidence implicating its contribution to the distinct pathogenesis resulting from HTLV-1 versus HTLV-2 infections.     

A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.     

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.     

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.     

Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant

High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity.