Usage of integrin and heparan sulfate as receptors for mouse adenovirus type 1.

Adenovirus fiber knobs are the capsid components that interact with binding receptors on cells, while an Arg-Gly-Asp (RGD) sequence usually found in the penton base protein is important for the interaction of most adenoviruses with integrin entry receptors. Mouse adenovirus type 1 (MAV-1) lacks an RGD sequence in the virion penton base protein. We tested whether an RGD sequence found in the MAV-1 fiber knob plays a role in infection. Treatment of cells with a competitor RGD peptide or a purified recombinant RGD-containing fiber knob prior to infection resulted in reduced virus yields compared to those of controls, indicating the importance of the RGD sequence for infection. An investigation of the role of integrins as possible receptors showed that MAV-1 yields were reduced in the presence of EDTA, an inhibitor of integrin binding, and in the presence of anti-alpha(v) integrin antibody. Moreover, mouse embryo fibroblasts that were genetically deficient in alpha(v) integrin yielded less virus, supporting the hypothesis that alpha(v) integrin is a likely receptor for MAV-1. We also investigated whether glycosaminoglycans play a role in MAV-1 infection. Preincubation of MAV-1 with heparin, a heparan sulfate glycosaminoglycan analog, resulted in a decrease in MAV-1 virus yields. Reduced MAV-1 infectivity was also found with cells that genetically lack heparan sulfate or cells that were treated with heparinase I. Cumulatively, our data demonstrate that the RGD sequence in the MAV-1 fiber knob plays a role in infection by MAV-1, alpha(v) integrin acts as a receptor for the virus, and cell surface heparin sulfate glycosaminoglycans are important in MAV-1 infection.

Heterocyclic compounds that inhibit Rev-RRE function and human immunodeficiency virus type 1 replication.

A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 muM concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.

Stem-loop IV in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication.

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.

Stem-loop III in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication.

Higher-order structures in the 5' untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5' UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of -9.1 kcal/mol in the positive strand and -3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that its associated intra-5' UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.