Stages Of Viral Life Cycle Research Articles

Antiviral mode of action of bovine dialyzable leukocyte extract against human immunodeficiency virus type 1 infection

BackgroundBovine dialyzable leukocyte extract (bDLE) is derived from immune leukocytes obtained from bovine spleen. DLE has demonstrated to reduce transcription of Human Immunodeficiency Virus Type 1 (HIV-1) and inactivate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Therefore, we decided to clarify the mode of antiviral action of bDLE on the inhibition of HIV-1 infection through a panel of antiviral assays.ResultsThe cytotoxicity, HIV-1 inhibition activity, residual infectivity of bDLE in HIV-1, time of addition experiments, fusion inhibition of bDLE for fusogenic cells and the duration of cell protection even after the removal of bDLE were all assessed in order to discover more about the mode of the antiviral action.HIV-1 infectivity was inhibited by bDLE at doses that were not cytotoxic for HeLa-CD4-LTR-β-gal cells. Pretreatment of HIV-1 with bDLE did not decrease the infectivity of these viral particles. Cell-based fusion assays helped to determine if bDLE could inhibit fusion of Env cells against CD4 cells by membrane fusion and this cell-based fusion was inhibited only when CD4 cells were treated with bDLE. Infection was inhibited in 80% compared with the positive (without EDL) at all viral life cycle stages in the time of addition experiments when bDLE was added at different time points. Finally, a cell-protection assay against HIV-1 infection by bDLE was performed after treating host cells with bDLE for 30 minutes and then removing them from treatment. From 0 to 7 hours after the bDLE was completely removed from the extracellular compartment, HIV-1 was then added to the host cells. The bDLE was found to protect the cells from HIV-1 infection, an effect that was retained for several hours.ConclusionsbDLE acted as an antiviral compound and prevented host cell infection by HIV-1 at all viral life cycle stages. These cell protection effects lingered for hours after the bDLE was removed. Interestingly, bDLE inhibited fusion of fusogenic cells by acting only on CD4 cells. bDLE had no virucidal effect, but could retain its antiviral effect on target cells after it was removed from the extracellular compartment, protecting the cells from infection for hours.bDLE, which has no reported side effects or toxicity in clinical trials, should therefore be further studied to determine its potential use as a therapeutic agent in HIV-1 infection therapy, in combination with known antiretrovirals.

The 2′,5′-Oligoadenylate Synthetase 1b Is a Potent Inhibitor of West Nile Virus Replication Inside Infected Cells

The 2',5'-oligoadenylate synthetase (OAS) proteins associated with endoribonuclease RNase L are components of the interferon-regulated OAS/RNase L system, which is an RNA decay pathway known to play an important role in the innate antiviral immunity. A large body of evidence suggests a critical role for the 1b isoform of the mouse Oas gene (Oas1b) in resistance to West Nile virus (WNV) infection in vivo. WNV is a positive, single-stranded RNA virus responsible for severe encephalitis in a large range of animal species and humans. To investigate the molecular basis for the sensitivity of WNV to the Oas1b antiviral pathway, we established a stable mouse fibroblastic cell clone that up-regulates Oas1b protein expression under the control of the Tet-Off expression system. We showed that murine cells respond to Oas1b expression by efficiently inhibiting WNV replication. The antiviral action of Oas1b was essentially restricted to the early stages in virus life cycle. We found that the inability of WNV to productively infect the Oas1b-expressing cells was attributable to a dramatic reduction in positive-stranded viral RNA level. Thus, Oas1b represents an antiviral pathway that exerts its inhibitory effect on WNV replication by preventing viral RNA accumulation inside infected cells.

Differential regulation of K8 gene expression in immediate-early and delayed-early stages of Kaposi's sarcoma-associated herpesvirus

KSHV-encoded bZip protein, namely K8, is a regulatory protein with multiple functions in immediate-early and delayed-early stages of viral life cycle. Here we report that K8 gene is expressed in both immediate-early and delayed-early phases and the transcription in different phases is controlled by distinct promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb. The transcription from the immediate-early promoter is inducible by sodium butyrate or 12- O-tetradecanoylphorbol-13-acetate (TPA), but not responsive to ORF50 (Rta). The delayed-early promoter of K8 shows little response to sodium butyrate and TPA, but can be fully induced by ORF50 (Rta). In the immediate-early promoter, a 20-bp region containing two consensus Sp-1 binding sites was found to be crucial for the basal transcription and sodium butyrate induction of the promoter. In addition, mutagenesis analyses identified three ORF50 (Rta) responsive elements (RREs) in the delayed-early promoter and their roles in ORF50-dependent transactivation were investigated. The differential regulation of K8 gene expression may represent a strategy in that the virus fine-tunes the levels of K8 protein in different stages or for distinct functions. The elucidation of K8 gene expression regulation helps in understanding roles of K8 in viral replication and pathogenicity.

Regulation of HIV replication by a host transcription factor NFκB

On the basis of sequence homology, four distinct groups of primate immunodeficiency viruses are now recognized. These viruses possess a number of auxiliary genes not found in other retroviruses, i.e., tat, rev, nef, vif, vpr, vpu, and vpx genes. The general conservation of these accessory genes, within a group and in some cases among all four primate immunodeficiency viruses, argl~es for an important functional role in the virus life cycle. These genes can be further characterized as essential and non-essential. Non-essential genes include nef, vif, vpr, vpu, and vpx. Human immunodeficiency virus type I (HIV-I), a magor causative agent of human AIDS, contains these auxiliary genes except for the vpx. Although the biology and molecular biology of HIV-I have been extensively studied, molecular bases for actions of the non-essential gene products, which are unique to the primate lentivirus group, are not fully understood yet. Recent studies have d~monstrated that most of the non-essential gene products act at various stages of virus life cycle in a cell-specific manner. We also have shown t_hat some gag gene mutants can replicate in some specific cell lines. The observed cell-specificity is critical for our understanding of the virology of HIV. In this report, to study functions of various HIV proteins, a large number of mutants were constructed, and bilogically and biochemically characterized. Growth kinetics of mutant viruses in several cell lines and primary blood cell cultures were determined, and defective steps in the viral life cycle of the mutants were monitored by our single-round replication assays. Target genes for study include gag, nef, vif, vpr, vpu, and vpx. We s~mmarize our new data regarding cell-dependent requirement of various HIV proteins for viral replication.