Mutational analysis of Turnip crinkle virus movement protein p8.

Summary Turnip crinkle virus encodes two proteins, p8 and p9, that are both required for cell-to-cell movement. The p8 movement protein has been demonstrated to bind RNA in a cooperative manner, although, similar to many other plant virus movement proteins, it contains no canonical RNA binding domain(s). However, three positively charged regions of p8 may potentially form ionic interactions with the RNA backbone. To identify functional regions of p8, a series of alanine and deletion scanning mutations were produced. The effects of these mutations were analysed using both in vitro RNA binding assays and in vivo infections of susceptible (Di-3) and resistant (Di-17) Arabidopsis thaliana plants. Several mutants that have reduced RNA binding ability were also demonstrated to be movement deficient and replication competent. Based on these results, there appear to be two regions, located between amino acids 18 and 31, and 50 and 72, that are required for RNA binding. Furthermore, additional regions (amino acids 12-15, and 34-37) appear to play a role in vivo unrelated to in vitro RNA binding activity.

The amino terminus of the coat protein of Turnip crinkle virus is the AVR factor recognized by resistant arabidopsis.

We have isolated three naturally occurring strains of Turnip crinkle virus (TCV) that break resistance in Di-17 Arabidopsis. Two mutations in the N terminus of the TCV coat protein, D4N and P5S, were shown to confer this phenotype. Thus, this region of the coat protein is involved in eliciting resistance responses in Arabidopsis.

A single amino acid change in turnip crinkle virus movement protein p8 affects RNA binding and virulence on Arabidopsis thaliana.

Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement protein p8 and inversely correlates with p8 RNA binding affinity.

Identification of an Arabidopsis locus required for resistance to turnip crinkle virus.

Inoculation of turnip crinkle virus (TCV) into a (TCV)-resistant line of Arabidopsis thaliana, Di-17, results in the development of a hypersensitive response (HR) on the inoculated leaves. In contrast, an HR does not occur when leaves of the TCV-susceptible Di-3 line or the susceptible ecotypes Columbia (Col-0), or Landsberg erecta (Ler) are inoculated. Genetic analysis of progeny from crosses between Di-17 and either Di-3, Col-0 or Ler demonstrates that the development of an HR is regulated by a single dominant nuclear locus, herein designated HRT. Using progeny from a Di-17 x Col-0 cross, HRT was mapped to chromosome 5, where it is tightly linked to the DFR locus. We also demonstrate that a variety of resistance-associated phenomena, including the TCV-induced accumulation of salicylic acid, camalexin and autofluorescent cell-wall material, correlate with the HR, suggesting the possibility that HRT is required for their activation.

Regulation of neighboring gene expression by the herpes simplex virus type 1 thymidine kinase gene.

The herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of the gH (UL22) gene with its 3' end overlapping the gH promoter, and it overlaps the UL24 gene's regulatory and coding sequences at its 5' end in a head-to-head orientation. Thus, tk expression could affect gH expression by promoter occlusion and UL24 expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects of tk promoter mutations that reduce tk expression on gH and UL24 expression. For gH, tk promoter mutations did not increase the accumulation of gH mRNA or the rate of gH transcription. Thus, tk expression does not appear to down-regulate gH expression. In contrast, we found that decreased tk expression correlated with increased accumulation of UL24 mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression of tk, but not at late times when tk mRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation of UL24 mRNA accumulation requires tk expression in cis. Nuclear run-off experiments did not detect differences in UL24 transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentially in cis.

Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia.

From marker rescue, sequencing, transcript, and latency analyses of the thymidine kinase-negative herpes simplex virus mutant dlsactk and studies using the thymidine kinase inhibitor Ro 31-5140, we infer that the virus-encoded thymidine kinase is required in murine trigeminal ganglia for acute replication and lytic gene expression, for increasing the numbers of cells expressing latency-associated transcripts, and for reactivation from latent infection.

Unusual regulation of expression of the herpes simplex virus DNA polymerase gene.

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.