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

VZV phylogeny.

<p>Phylogenetic network (splits network) of available complete genome VZV sequences. The strains are divided into clades 1 to 5, and the deep lineage reticulate topology is caused by a few ancient recombination events described previously. The network also presents a reticulate pattern within clade 1 caused by a putative recent recombination event involving strain SVETA.</p

HSV-1 timescale estimation based on the genes US7 and US8.

<p>Estimated timescale for the HSV-1 evolution. Each major branch point in the phylogenetic tree is marked with a dotted line to the scale-bar below. The 95% confidence intervals for each predicted time since divergence are denoted with purple bars. The increased number of branch points in each clade is highlighted with green background. Posterior probabilities for the major clades are shown.</p

HSV-1 recombination analysis.

<p>Recombination analysis using Bootscan and SimPlot. A and B illustrate the bootscan and SimPlot analysis of query sequences 2762 and 4-J1037, respectively, which were classified as non-recombinants based on the US7–US8 regions. Bootscan plots demonstrate highly fragmented genomes as a result of recombination. Similarity plots demonstrate the sequence similarity between the query sequence and the other sequences. C and D depicts the bootscan analysis of the reference strains F and 17. Nucleotide positions refer to the sequence alignment excluding gaps and repeat regions.</p

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

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

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

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