Quantification of the Host Response Proteome after Herpes Simplex Virus Type 1 Infection

Viruses employ numerous host cell metabolic functions to propagate and manage to evade the host immune system. For herpes simplex virus type 1 (HSV1), a virus that has evolved to efficiently infect humans without seriously harming the host in most cases, the virus–host interaction is specifically interesting. This interaction can be best characterized by studying the proteomic changes that occur in the host during infection. Previous studies have been successful at identifying numerous host proteins that play important roles in HSV infection; however, there is still much that we do not know. This study identifies host metabolic functions and proteins that play roles in HSV infection, using global quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling of the host cell combined with LC–MS/MS. We showed differential proteins during early, mid and late infection, using both cytosolic and nuclear fractions. We identified hundreds of differentially regulated proteins involved in fundamental cellular functions, including gene expression, DNA replication, inflammatory response, cell movement, cell death, and RNA post-transcriptional modification. Novel differentially regulated proteins in HSV infections include some previously identified in other virus systems, as well as fusion protein, involved in malignant liposarcoma (FUS) and hypoxia up-regulated 1 protein precursor (HYOU1), which have not been identified previously in any virus infection

Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify <i>host</i> proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34­(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells

Influenza A Infection of Primary Human Airway Epithelial Cells Up-Regulates Proteins Related to Purine Metabolism and Ubiquitin-Related Signaling

Virus–host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC–MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy

Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify <i>host</i> proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34­(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells

Influenza A Infection of Primary Human Airway Epithelial Cells Up-Regulates Proteins Related to Purine Metabolism and Ubiquitin-Related Signaling

Virus–host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC–MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy

Quantification of the Host Response Proteome after Mammalian Reovirus T1L Infection

<div><p>All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular “transcriptome.” We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.</p> </div

Kinetics of reovirus growth and viral-induced cytopathology.

<p>Each of five different cell lines (L929, A549, HEK293, CaCo₂ and Hela) were infected at MOI  = 1 PFU/cell with T1L (a) or T3D (b). Cell lysates were harvested at 0, 24, 48 and 72hpi and titrated. Experiments were performed in triplicate; error bars represent standard error. Virus titers were greatest in the L929 and HEK293 cells for both virus strains. HEK293 (c) and L929 (d) cells were then re-analyzed as in (a) and (b) after infection at MOI  = 5 and at additional time points. Aliquots of the infections in (c) and (d) were also assessed for cell viability by trypan blue exclusion (e and f, respectively), with 100 μg/ml puromycin used as a positive cell killing control. Experiments were performed in duplicate; error bars represent standard error.</p

Top network functions generated using Ingenuity protein analysis for HEK293 cells infected with T1L reovirus at (a) 6hpi and (b) 24hpi.

<p>Graphs represent host cell functions with highest score (x-axis) based on the number of differentially regulated proteins observed in that network. The higher the score, the greater the number of proteins differentially regulated in that particular function network.</p

HEK293 proteins decreased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p

HEK293 proteins increased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p