The expression of the genomic information of severe acute respiratory syndrome coronavirus (SARS CoV) involves synthesis of a nested set of subgenomic RNAs (sgRNAs) by discontinuous transcription. In ARS CoV-infected cells, 10 sgRNAs, including 2 novel ones, were identified, which were predicted to be functional in the expression of 12 open reading frames located in the 3 one-third of the genome. Surprisingly, one new sgRNA could lead to production of a truncated spike protein. Sequence analysis of the leader-body fusion sites of each sgRNA showed that the junction sequences and the orresponding transcription-regulatory sequence (TRS) are unique for each species of sgRNA and are consistent after virus passages. For the two novel sgRNAs, each used a variant of the TRS that has one nucleotide mismatch in the conserved hexanucleotide core (ACGAAC) in the TRS. Coexistence of both plus and minus strands of SARS CoV sgRNAs and evidence for derivation of the sgRNA core sequence from the body core sequence favor the model of discontinuous transcription during minus-strand synthesis. Moreover, one rare species of sgRNA has the junction sequence AAA, indicating that its transcription could result from a noncanonical transcription signal. Taken together, these results provide more insight into the molecular mechanisms of genome expression and subgenomic transcription of SARS CoV.

RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. We demonstrated that intracellular expression of short hairpin RNA (shRNA) targeting human cyclin T1 (hCycT1), a cellular factor essential for transcription of messenger and genomic RNAs from the long terminal repeat promoter of provirus of human immunodeficiency virus type 1 (HIV-1), could effectively suppress the replication of HIV-1. We also showed that downregulation of hCycT1 did not cause apoptotic cell death, therefore, targeting cellular factor hCycT1 by shRNAs may provide an attractive approach for genetic therapy of HIV-1 infection in the future.

We used a bovine brain cDNA library to perform a yeast two-hybrid assay with bovine mature PrPC as bait. The screening result showed that aB-crystalline interacted with PrPC. The interaction was further evaluated both in vivo and in vitro with different methods, such as immunofluorescent colocalization, native polyacrylamide-gel electrophoresis, and IAsys biosensor assays. The results suggested that aB-crystalline may have the ability to refold denatured prion proteins, and provided first evidence that aB-crystalline is directly associated with prion protein.

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA)(genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.