Temperature protects insect cells from infection by cricket paralysis virus.

Heat shock is a well-known stress response characterized by a rapid synthesis of a set of proteins which are responsible for protection against stress. We examined the role of temperature on the growth of cricket paralysis virus, a member of the family Dicistroviridae, in insect cells. Heat shock caused an induction of heat shock protein-encoding mRNAs in uninfected cells but not in infected cells. While viral RNA and protein were abundant during heat shock, virion formation was inhibited at higher temperatures. The different susceptibility to pathogens at different temperatures is likely a crucial feature of host-pathogen interaction in cold-blooded animals.

An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.

Cellular stress such as endoplasmic reticulum stress, hypoxia, and viral infection activates an integrated stress response, which includes the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit overall protein synthesis. Paradoxically, this leads to translation of a subset of mRNAs, like transcription factor ATF4, which in turn induces transcription of downstream stress-induced genes such as growth arrest DNA-inducible gene 34 (GADD34). GADD34 interacts with protein phosphatase 1 to dephosphorylate eIF2alpha, resulting in a negative feedback loop to recover protein synthesis and allow translation of stress-induced transcripts. Here, we show that GADD34 is not only transcriptionally induced but also translationally regulated to ensure maximal expression during eIF2alpha phosphorylation. GADD34 mRNAs are preferentially associated with polysomes during eIF2alpha phosphorylation, which is mediated by its 5'-untranslated region (5'UTR). The human GADD34 5'UTR contains two non-overlapping upstream open reading frames (uORFs), whereas the mouse version contains two overlapping and out of frame uORFs. Using 5'UTR GADD34 reporter constructs, we show that the downstream uORF mediates repression of basal translation and directs translation during eIF2alpha phosphorylation. Furthermore, we show that the upstream uORF is poorly translated and that a proportion of scanning ribosomes bypasses the upstream uORF to recognize the downstream uORF. These findings suggest that GADD34 translation is regulated by a unique 5'UTR uORF mechanism to ensure proper GADD34 expression during eIF2alpha phosphorylation. This mechanism may serve as a model for understanding how other 5'UTR uORF-containing mRNAs are regulated during cellular stress.

Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp.

The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

Immunodominance among herpes simplex virus-specific CD8 T cells expressing a tissue-specific homing receptor.

The study of immunodominance within microbe-specific CD8 T cell responses has been challenging. We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides. Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen. The specificity of the resultant cytotoxic clones was determined. Clonal frequencies were compared with each other and with the total number of cytotoxic clones. For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides. Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects. Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots. We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.