Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production.

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important cofactors that promote L-protein stability and RNA synthesis.

UNLABELLED: The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE: Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.

Mapping of CD8 T cell epitopes in human respiratory syncytial virus L protein.

OBJECTIVES: Since it has been reported that in humans there is a relationship between human respiratory syncytial virus (hRSV)-specific cytotoxic T lymphocytes and symptom reduction, and that the polymerase (structural L protein) is highly conserved among different strains, this work aimed to identify the CD8 T cell epitopes H-2(d) restricted within the L sequence for immunization purposes. METHODS: We screened the hRSV strain A2 L protein sequence using two independent algorithms, SYFPEITHI and PRED/(BALB/c), to predict CD8 T cell epitopes. The selected peptides were synthesized and used to immunize BALB/c mice for the evaluation of T cell response. The production of IFN-γ from splenocytes of hRSV-infected animals stimulated by these peptides was assayed by ELISPOT. RESULTS: Nine peptides showing the best binding scores to the BALB/c MHC-I molecules (H-2K(d), L(d) and D(d)) were selected. Sequence homology analysis showed that these sequences are conserved among different hRSV strains. Two of these peptides induced significant IFN-γ production by ex vivo-stimulated T cells. CONCLUSIONS: Our results indicate that the hRSV L protein contains H-2(d)-restricted epitopes.

Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant.

Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells.

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.

Structural analysis of human respiratory syncytial virus p protein: identification of intrinsically disordered domains.

Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice.

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (P) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines.

Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system.

OBJECTIVES: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. METHODS: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. RESULTS: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. CONCLUSIONS: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant.

Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli.

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.

Nitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cells.

The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.

Polymerase chain reaction detection of adenovirus DNA sequences in human lymphocytes

Seqüências de DNA de adenovirus são freqüentemente encontradas em linfócitos humanos, tendo sido utilizadas para tal as técnicas de PCR e "Southern blot". Isto é possível apesar dessas células não serem permissivas à replicação de adenovírus, sugerindo persistência do genoma viral. Para investigar esse fenômeno, procuramos detectar em voluntários não sintomáticos DNA adenoviral e expressão do gene E1A. Amostras de DNA obtidas de glóbulos brancos periféricos de 51 voluntários, foram submetidas à PCR utilizando oligonucleotídeos para uma seqüência conservada do gene hexon, seguindo-se uma "nested PCR". Seqüências de adenovirus foram encontradas em 27 amostras (52,9 per center). Depois de mais de um ano, novas amostras desses voluntários positivos foram analisadas e em 70,8 per center dos casos o resultado foi mantido. Como isto poderia ser devido à persistência, decidimos verificar se o gene precoce E1A estava relacionado analisando sua expressão através de RT-PCR. Os resultados foram negativos para todas as amostras. O par de oligonucleotídeos desenvolvido para essa finalidade, que tem como alvo uma região conservada em E1A, permitiu detectar seqüências de adenovírus diretamente por PCR. A concordância encontrada entre essa análise e a pesquisa para o gene hexon foi de 84 per center. Nossos dados sugerem alta ocorrência e persistência de genoma adenoviral em linfócitos humanos, e indicam que uma região distinta de E1A é responsável pela persistência. Podemos também afirmar que a PCR para o gene E1A é uma boa opção quando se quer detectar adenovírus, evitando-se o alto risco de contaminação da "nested PCR" necessária para identificar a presença do gene hexon.