The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein.

The membrane (M1) protein of influenza virus was found to be heterogenous and to occur in two forms in the virus particle. The two forms of M1 were found in virus which was produced both early and late after infection and in infected cells. The two forms could be separated on polyacrylamide gels under specific conditions. The two components of M1 contained similar tryptic peptides. However, a small proteolytic difference between the two proteins could not be ruled out. Both M1 proteins were present in phosphorylated form in the virus particle. The phosphorylated M1 components were not readily distinguished from phosphorylated nonstructural protein (NS1) when cytoplasm of infected cells was analyzed on polyacrylamide gels. The two phosphorylated M1 components could, however, be detected in infected cells by immunoprecipitation. One M1 component contained only phosphoserine whereas the second contained phosphoserine and a small amount of phosphothreonine as well. In addition to the phosphorylated nucleoprotein and M1, a third phosphorylated protein was routinely detected in virus particles. It was a surface component of the virus, since it could be removed from whole virus with chymotrypsin and contained phosphate at serine residues. The identity of this component was not known.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [³⁵S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Rotavirus infection induces the phosphorylation of eIF2alpha but prevents the formation of stress granules

Early during the infection process, rotavirus causes the shutoff of cell protein synthesis, with the nonstructural viral protein NSP3 playing a vital role in the phenomenon. In this work, we have found that the translation initiation factor 2α (eIF2α) in infected cells becomes phosphorylated early after virus infection and remains in this state throughout the virus replication cycle, leading to a further inhibition of cell protein synthesis. Under these restrictive conditions, however, the viral proteins and some cellular proteins are efficiently translated. The phosphorylation of eIF2α was shown to depend on the synthesis of three viral proteins, VP2, NSP2, and NSP5, since in cells in which the expression of any of these three proteins was knocked down by RNA interference, the translation factor was not phosphorylated. The modification of this factor is, however, not needed for the replication of the virus, since mutant cells that produce a nonphosphorylatable eIF2α sustained virus replication as efficiently as wild-type cells. In uninfected cells, the phosphorylation of eIF2α induces the formation of stress granules, aggregates of stalled translation complexes that prevent the translation of mRNAs. In rotavirus-infected cells, even though eIF2α is phosphorylated these granules are not formed, suggesting that the virus prevents the assembly of these structures to allow the translation of its mRNAs. Under these conditions, some of the cellular proteins that form part of these structures were found to change their intracellular localization, with some of them having dramatic changes, like the poly(A) binding protein, which relocates from the cytoplasm to the nucleus in infected cells, a relocation that depends on the viral protein NSP3.     

Preferential Phosphorylation of NP-Protein of Influenza A2 Virus by Virion-Associated Protein Kinase

Influenza A₂ virions were found to contain protein kinase activity which was stimulated, like in other virion-associated kinases, with Mg⁺⁺ and Nonidet-P 40 but not with cyclic AMP. The kinase phosphorylated only the NP-protein fraction of the influenza virions in the in vitro reaction. In contrast, none of the influenza virion proteins were phosphorylated significantly during the process of virus production in infected chorioallantoic membranes, The in vitro and in vivo phosphorylations of influenza viral proteins were compared with those of Sendai virus (HVJ).     

Complementation of translational defect for growth of human adenovirus type 2 in Simian cells by a Simian virus 40-induced factor

The defective step which leads human adenovirus type 2 infection of African green monkey kidney cells (clone C14) to be abortive and its complementation in simian virus 40-transformed cells (clone T22) were studied by comparing the synthesis and function of macromolecules in these cell lines. Neither a quantitative nor a qualitative difference was detected in virus DNA replication and in virus mRNA synthesis in these cells, while a definite difference was observed in protein synthesis. The capsid proteins, such as hexon or penton, were synthesized in T22 cells but not in C14 cells. Inability of polyribosomes to synthesize the capsid proteins in C14 cells infected with adenovirus type 2 may not be due to a defect in elongation of nascent polypeptides or their release, since nascent polypeptides pulse-labelled with [³H]leucine were completely released from polyribosomes after the chase. The electrophoretic analysis of proteins synthesized in vitro with polyribosomes from either infected T22 or C14 cells using the pH 5 enzyme and S100 fraction from T22 cells revealed that hexon was synthesized with polyribosomes from T22 cells but not from C14 cells, thereby suggesting that the defect is not ascribed to a component in the pH 5 enzyme and S100 fraction, but resides in polyribosomes. The analysis of late adenovirus mRNA associated with polyribosomes in the infected T22 and C14 cells by hybridization competition or by sedimentation revealed that all the species of virus mRNA were present in the cytoplasm of these cells; however, certain species of virus mRNA larger than 20 S were absent in polyribosomes of the infected C14 cells. Sedimentation analysis of late adenovirus mRNA following separation on poly(U)-Sepharose or by membrane filtration gave the same results. These results suggest that the defect of C14 cells to support growth of adenoviruses is due to the inability of ribosomes to associate with certain species of late virus mRNA to form polyribosomes and suggest that a factor complementing this defect is induced by simian virus 40.     

Evasion of Antiviral Immunity through Sequestering of TBK1/IKKε/IRF3 into Viral Inclusion Bodies

Cells are equipped with pattern recognition receptors (PRRs) such as the Toll-like and RIG-I-like receptors that mount innate defenses against viruses. However, viruses have evolved multiple strategies to evade or thwart host antiviral responses. Viral inclusion bodies (IBs), which are accumulated aggregates of viral proteins, are commonly formed during the replication of some viruses in infected cells, but their role in viral immune evasion has rarely been explored. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus in the Bunyaviridae. The SFTS viral nonstructural protein NSs can suppress host beta interferon (IFN-β) responses. NSs can form IBs in infected and transfected cells. Through interaction with tank-binding kinase 1 (TBK1), viral NSs was able to sequester the IKK complex, including IKKε and IRF3, into IBs, although NSs did not interact with IKKε or IRF3 directly. When cells were infected with influenza A virus, IRF3 was phosphorylated and active phosphorylated IRF3 (p-IRF3) was translocated into the nucleus. In the presence of NSs, IRF3 could still be phosphorylated, but p-IRF3 was trapped in cytoplasmic IBs, resulting in reduced IFN-β induction and enhanced viral replication. Sequestration of the IKK complex and active IRF3 into viral IBs through the interaction of NSs and TBK1 is a novel mechanism for viral evasion of innate immunity.     

Polyomavirus-Associated Trichodysplasia Spinulosa Involves Hyperproliferation,pRB Phosphorylation and Upregulation of p16 and p21

Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16^ink4a, p21^waf, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16^ink4a and p21^waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Patients Infected with CRF07_BC Have Significantly Lower Viral Loads than Patients with HIV-1 Subtype B: Mechanism and Impact on Disease Progression

The circulating recombinant form (CRF) 07_BC is the most prevalent HIV-1 strain among injection drug users (IDUs) in Taiwan. It contains a 7 amino-acid deletion in its p6^gag. We conducted a cohort study to compare viral loads and CD4 cell count changes between patients infected with subtype B and CRF07_BC and to elucidate its mechanism. Twenty-one patients infected with CRF07_BC and 59 patients with subtype B were selected from a cohort of 667 HIV-1/AIDS patients whom have been followed up for 3 years. Generalized estimated equation was used to analyze their clinical data and the results showed that patients infected with CRF07_BC had significantly lower viral loads (about 58,000 copies per ml less) than patients with subtype B infection (p = 0.002). The replicative capacity of nine CRF07_BC and four subtype B isolates were compared and the results showed that the former had significantly lower replicative capacity than the latter although all of them were CCR5- tropic and non-syncytium inducing viruses. An HIV-1-NL4-3 mutant virus which contains a 7 amino-acid deletion in p6^gag (designated as 7d virus) was generated and its live cycle was investigated. The results showed that 7d virus had significantly lower replication capacity, poorer protease-mediated processing and viral proteins production. Electron microscopic examination of cells infected with wild-type or 7d virus demonstrated that the 7d virus had poorer and slower viral maturation processes: more viruses attached to the cell membrane and higher proportion of immature virions outside the cells. The interaction between p6^gag and Alix protein was less efficient in cells infected with 7d virus. In conclusion, patients infected with CRF07_BC had significantly lower viral loads than patients infected with subtype B and it may due to the deletion of 7 amino acids which overlaps with Alix protein-binding domain of the p6^gag.     

Interactions of Vesicular Stomatitis Virus Structural Proteins with HeLa Plasma Membranes

THE processes whereby nucleoprotein core particles of certain animal viruses become enveloped by and bud off from host cell membranes can be studied by preparing membrane^1,2 or “sedimentable”³ fractions from infected cells and examining them for the presence of virus proteins. We find that similar experiments designed to monitor assembly of vesicular stoma-titus virus (VSV) at sites along the plasma membranes of HeLa cells are best interpreted after first investigating the possibility that virus proteins adsorb to plasma membranes during cell fractionation and membrane isolation. In this report, we show that at 0° C the membrane protein of VSV, among other virus proteins, adsorbs to plasma membranes isolated from uninfected HeLa cells. With appropriate pulse-chase experiments, however, we are able to demonstrate the progressive association, in vivo, of VSV core protein with plasma membranes of infected HeLa cells.     

Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells.

Recombinant baculoviruses were constructed which express simian virus 40 large T antigen (SVT-Ag) or murine p53 to high levels in infected insect cells. Characterization of the expressed proteins revealed that they display many properties of the corresponding mammalian-derived proteins. Both proteins are of wild-type size, localize to the nucleus, are recognized by several SVT-Ag- or p53-specific monoclonal antibodies, and are phosphorylated in this system. Complexes are formed between baculovirus-derived SVT-Ag and p53 after coinfection of insect cells with both recombinant viruses. After infection of insect cells with either virus individually, each protein can self-associate to form a variety of oligomeric species. Pulse-chase experiments indicated that both SVT-Ag and p53 are highly stable in insect cells, even in the absence of complex formation.     

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M_r, termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33°C. Anti-C3 serum specifically recognized [³H]leucine-labeled P85, but not P58, from infected cells maintained at 33°C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with ³²P_i showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected ³²P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63^gag. Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63^gag. We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85^gag-mos. We have not detected any other v-mos gene product in ts110-infected cells.     

Interferon-induced synthesis of a 63,000 dalton protein in mouse cells.

Cultures of mouse JLSV5 cells (a cell line chronically infected with Rauscher murine leukemia virus) and of fresh uninfected NMRI mouse embryo fibroblasts were treated with interferon and labelled with (³⁵S)-methionine. Newly synthesized proteins were then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cell lysates. In both cell types interferon treatment resulted in the synthesis of a radioactive 63,000 dalton protein, which was undetectable in a radioactive form in the control cells. The possibility is considered that this protein is a mediator of the biological activities of interferon on cells.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

Analysis of human peripheral blood samples from fatal and nonfatal cases of Ebola (Sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels

Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8⁺ T cells, and activated (HLA-DR⁺) CD8⁺ T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (≥150 μM) would have negatively affected vascular tone and contributed to virus-induced shock.