Preparation of New Sargassum fusiforme Polysaccharide Long-Chain Alkyl Group Nanomicelles and Their Antiviral Properties against ALV-J.

Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus which has caused heavy losses to the poultry breeding industry. Currently, there is no effective medicine to treat this virus. In our previous experiments, the low-molecular-weight Sargassum fusiforme polysaccharide (SFP) was proven to possess antiviral activity against ALV-J, but its function was limited to the virus adsorption stage. In order to improve the antiviral activity of the SFP, in this study, three new SFP long-chain alkyl group nanomicelles (SFP-C12M, SFP-C14M and SFP-C16M) were prepared. The nanomicelles were characterized according to their physical and chemical properties. The nanomicelles were characterized by particle size, zeta potential, polydispersity index, critical micelle concentration and morphology. The results showed the particle sizes of the three nanomicelles were all approximately 200 nm and SFP-C14M and SFP-C16M were more stable than SFP-C12M. The newly prepared nanomicelles exhibited a better anti-ALV-J activity than the SFP, with SFP-C16M exhibiting the best antiviral effects in both the virus adsorption stage and the replication stage. The results of the giant unilamellar vesicle exposure experiment demonstrated that the new virucidal effect of the nanomicelles might be caused by damage to the phospholipid membrane of ALV-J. This study provides a potential idea for ALV-J prevention and development of other antiviral drugs.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

A novel Bursin-like peptide as a potential virus inhibitor and immunity regulator in SPF chickens infected with recombinant ALV.

BACKGROUND: Avian leukosis viruses (ALVs) are important contagious suppressive factors of chicken immunity and growth performance, resulted in enormous economic loss. Although virus eradication programs are applied in breeder flocks, ALVs are still widespread globally. Therefore, other valuable adjunct to reduce the negative effect of ALVs should be considered. Bursin-like peptide (BLP) showed remarkable immunomodulatory effects, whereas their influence on ALV-infected avian groups has not been reported. Here, a designed hybrid BLP was expressed in E. coli. The purified BLP was injected subcutaneously weekly in SPF chickens congenitally infected with a natural ALV strain. Then the influences of this BLP on the growth performance, immune response and virus titer of ALV-infected chickens were determined. RESULTS: This BLP injection significantly improved the body weights of ALV-infected birds (P < 0.05). BLP injection significantly enhanced organ index in the BF in ALV-infected birds (P < 0.05). The weekly injection of BLP significantly lengthened the maintenance time of antibodies against Newcastle disease virus (NDV) attenuated vaccine of ALV-infected birds (P < 0.05) and boosted the antibody titer against avian influenza virus (AIV) H5 inactive vaccine of mock chicken (P < 0.05). BLP injection in mock chickens enhanced the levels of serum cytokines (IL-2, IL-4 and interferon-γ) (P < 0.05). Surprisingly, the novel BLP significantly inhibited expression of the ALV gp85 gene in the thymus (P < 0.05), kidney (P < 0.05) and bursa of Fabricius (BF) (P < 0.01) of ALV-infected chickens. Both viral RNA copy number and protein level decreased significantly with BLP (50 µg/mL) inoculation before ALV infection in DF1 cells (P < 0.05). CONCLUSIONS: This is the first report investigating the influence of BLP on the growth and immunity performance of chickens infected by ALV. It also is the first report about the antiviral effect of BLP in vivo and in vitro. This BLP expressed in E. coli showed potential as a vaccine adjuvant, growth regulator and antiretroviral drug in chickens to decrease the negative effects of ALV infection.

Taishan Pinus Massoniana pollen polysaccharide inhibits the replication of acute tumorigenic ALV-J and its associated tumor growth.

Avian leukosis virus subgroup J (ALV-J) has resulted in considerable economic losses in the poultry industry. In recent years, fibrosarcoma induced by ALV-J, which contains the v-fps oncogene, has gained momentum, and this has brought about new challenges to the poultry industry. To study the inhibitory effects of Taishan Pinus Massoniana pollen polysaccharide (TPPPS) on acute ALV-J infection and tumor development, antiviral and antitumor models of the Fu-J (SDAU1005) strain of ALV-J were established in vitro and in vivo. The results of in vitro experiments showed that TPPPS significantly inhibited viral replication in a dose-dependent manner during adsorption and pretreatment stages. The results of in vivo experiments have shown that TPPPS significantly reduced the viral load in the plasma and tumor tissues, as well as inhibited tumor growth. We further examined the difference in transcriptome expression by using RNA-Seq technology. A total of 560 differentially expressed genes were identified that included 329 up-regulated genes and 231 down-regulated genes. The up-regulated genes were mainly immune-related genes, whereas the down-regulated genes were mainly tumor-regulated genes. Gene Ontology (GO) term enrichment included immune system processes, positive regulation of immune system processes, regulation of immune system processes, leukocyte activation, cell activation, and protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the main immune and tumor-related pathways included T-cell receptor signaling pathway, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, and Ras signaling pathway. In summary, our results preliminarily point to the antiviral and antitumor mechanism of TPPPS in vivo and in vitro.

The antiviral property of Sargassum fusiforme polysaccharide for avian leukosis virus subgroup J in vitro and in vivo.

Avian Leukosis Virus Subgroup J (ALV-J) is an oncogenic retrovirus, mainly spread by vertical and horizontal transmission, which have caused severe losses in world poultry industry. Sargassum fusiforme polysaccharide (SFP), a marine algae sulfated polysaccharide, has attracted more attention due to its variously biological activities. In this study, the anti-ALV-J property of SFP was assessed in vivo and in vitro. The results demonstrated that different Mw of SFPs showed virustatic activity to ALV-J in vitro by combining with the virus when ALV-J adsorbed onto the host cells. When treated with SFPs, the ALV-J gene and protein expression reduced clearly and SFP-3 (Molecular weight 9 kDa) had the best antiviral effect. Results in vivo showed that the immunosuppression of the ALV-J infected chickens were relieved by SFP-3. Moreover, SFP-3 obviously inhibit the viral shedding and alleviated the organs damage caused by ALV-J. This study offered a new method for ALV-J treatment and enriched the potential application of SFP.

Joint treatment with azidothymidine and antiserum for eradication of avian leukosis virus subgroup a contamination in vaccine virus seeds.

Contamination of avian attenuated vaccines by avian leukosis virus (ALV) is considered to be a specific horizontal transmission of ALV. Eradication of ALV contamination in vaccine virus seeds is thus a precondition of qualified vaccine production. In this study, we used the nucleoside reverse transcriptase (RT) inhibitor azidothymidine (AZT) together with monofactor antiserum against avian leukosis virus subgroup A (ALV-A) to remove ALV-A from vaccine virus seeds. Different doses of ALV-A were artificially added to the Newcastle disease virus (NDV) vaccine seeds, then the ALV-contaminated attenuated virus vaccine virus seeds were cultured in DF-1 cells. Single-drug treatment with 5 µg/mL AZT or 5% (v/v) as well as combined treatment with AZT and antiserum significantly suppressed ALV-A replication (P < 0.001) in the vaccine virus seeds. Complete absence of virus replication was observed in cells exposed to joint treatment with AZT and antiserum. The treated virus seeds met the requirements of the Chinese Ministry of Agriculture. Therefore, combined treatment with AZT and antiserum can be used to eradicate contaminating ALV-A from vaccine virus seeds, thus providing a new approach for improving vaccine safety.

Antiviral Activity against Avian Leucosis Virus Subgroup J of Degraded Polysaccharides from Ulva pertusa.

Avian Leukosis Virus Subgroup J (ALV-J), a retrovirus of avian, has caused enormous economics losses to poultry industry around the world. Polysaccharides from marine algae are featured diversity bioactivities. To find the potential effect to prevent ALV-J spread, in this study, polysaccharides from Ulva pertusa (UPPs) and four low molecular weight (Mw) U. pertusa polysaccharides (LUPPs) were prepared and their functions on ALV-J were investigated. Firstly, LUPPs were obtained by hydrogen peroxide (H2O2) oxidative degradation. The effects of degradation conditions on Mw of the UPP were also investigated. Results showed that the H2O2 oxidative degradation method could degrade UPP effectively, and the degradation was positively related to H2O2 concentration and temperature and negatively to pH. The chemical characteristics of UPP and LUPPs were also determined. Afterwards, the anti-ALV-J activity of the polysaccharides were carried out in vitro. Results showed that UPP and LUPPs could inhibit ALV-J and LUPP-3 and Mw of 4.3 kDa exerted the strongest suppression. The action phase assay showed that LUPP-3 could bind with the viral particles and prevented ALV-J adsorption onto the host cells. And the ALV-J relative gene and gp85 protein expression were significantly suppressed after being administration with LUPP-3. Therefore, the low Mw polysaccharides from U. pertusa have great potential as an anti-ALV-J drug alternative.

Baicalin is an inhibitor of subgroup J avian leukosis virus infection.

Avian leukosis virus subgroup J (ALV-J) can cause great economic losses to the poultry industry worldwide. Baicalin, one of the flavonoids present in S.baicalensis Georgi, has been shown to have antiviral activities. To investigate whether baicalin has antiviral effects on the infection of ALV-J in DF-1 cells, the cells were treated with baicalin at different time points. We found that baicalin could inhibit viral mRNA, protein levels and overall virus infection in a dose- and time-dependent manner using a variety of assays. Baicalin specifically targeted virus internalization and reduced the infectivity of ALV-J particles, but had no effect on the levels of major ALV-J receptor and virus binding to DF-1 cells. Collectively, these results suggest that baicalin might have potential to be developed as a novel antiviral agent for ALV-J infection.

Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture.

Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.

Degradation of Polysaccharides from Grateloupia filicina and Their Antiviral Activity to Avian Leucosis Virus Subgroup J.

In this study, polysaccharides from Grateloupia filicinia (GFP) were extracted and several low molecular weight (Mw) G. filicina polysaccharides (LGFPs) were prepared by the hydrogen peroxide (H2O2) oxidation method. Additionally, the effect of different experimental conditions on the degradation of GFP was determined. Results showed that the GFP degradation rate was positively related to H2O2 concentration and temperature, and negatively related to pH. Chemical analysis and Fourier transform infrared spectra (FT-IR) of GFP and LGFPs showed that the degradation caused a slight decrease of total sugar and sulfate content. However, there was no obvious change for monosaccharide contents. Then, the anti-ALV-J activity of GFP and LGFPs were determined in vitro. Results revealed that all of the samples could significantly inhibit ALV-J and lower Mw LGFPs exhibited a stronger suppression, and that the fraction LGFP-3 with Mw 8.7 kDa had the best effect. In addition, the reaction phase assays showed that the inhibition effect was mainly because of the blocking virus adsorption to host cells. Moreover, real-time PCR, western-blot, and IFA were further applied to evaluate the blocking effects of LGFP-3. Results showed that the gene relative expression and gp85 protein for LGFPS-3 groups were all reduced. Data from IFA showed that there was less virus infected cells for 1000 and 200 µg/mL LGFPS-3 groups when compared to virus control. Therefore, lower Mw polysaccharides from G. filicina might supply a good choice for ALV-J prevention and treatment.

Taishan Pinus massoniana pollen polysaccharide inhibits subgroup J avian leucosis virus infection by directly blocking virus infection and improving immunity.

Subgroup J avian leucosis virus (ALV-J) generally causes neoplastic diseases, immunosuppression and subsequently increases susceptibility to secondary infection in birds. The spread of ALV-J mainly depends on congenital infection and horizontal contact. Although ALV-J infection causes enormous losses yearly in the poultry industry worldwide, effective measures to control ALV-J remain lacking. In this study, we demonstrated that Taishan Pinus massoniana pollen polysaccharide (TPPPS), a natural polysaccharide extracted from Taishan Pinus massoniana pollen, can significantly inhibit ALV-J replication in vitro by blocking viral adsorption to host cells. Electron microscopy and blocking ELISA tests revealed that TPPPS possibly blocks viral adsorption to host cells by interacting with the glycoprotein 85 protein of ALV-J. Furthermore, we artificially established a congenitally ALV-J-infected chicken model to examine the anti-viral effects of TPPPS in vivo. TPPPS significantly inhibited viral shedding and viral loads in immune organs and largely eliminated the immunosuppression caused by congenital ALV-J infection. Additionally, pre-administration of TPPPS obviously reduced the size and delayed the occurrence of tumors induced by acute oncogenic ALV-J infection. This study revealed the prominent effects and feasible mechanisms of TPPPS in inhibiting ALV-J infection, thereby providing a novel prospect to control ALV-J spread.

Efficacy of an autophagy-targeted DNA vaccine against avian leukosis virus subgroup J.

Infection with the avian leukosis virus subgroup J (ALV-J) can lead to neoplastic disease in chickens, inflicting significant economic losses to the poultry industry. Recent reports have identified inhibitory effects of ALV-J on autophagy, a process involving in innate and adaptive immunity. Inspired by this connection between autophagy and immunity, we developed a novel DNA vaccine against ALV-J which includes co-administration of rapamycin to stimulate autophagy. To measure the efficacy of the developed prototype vaccine, five experimental groups of seven-day-old chickens was immunized three times at three-week intervals respectively with vector, pVAX1-gp85, pVAX1-gp85-LC3, pVAX1-gp85+rapamycin and pVAX1-gp85-LC3+rapamycin through electroporation. We then tested their antibody titers, cytokine levels and cellular immune responses. The immunoprotective efficacy of the prototype vaccines against the challenge of the ALV-J GD1109 strain was also examined. The results showed that the combination of pVAX1-gp85-LC3 and rapamycin was able to induce the highest antibody titers, and enhance interleukin(IL)-2, IL-10 and interferon (IFN)-γ expression, and the chickens immunized with the combination of pVAX1-gp85-LC3 and rapamycin showed the highest percentage of CD3+CD8+T lymphocytes. Based on our results, we suggest that stimulating autophagy can improve the efficacy of DNA vaccines and that our DNA vaccine shows the potential of being a candidate vaccine against ALV-J. This study provides a novel strategy for developing vaccines against ALV-J.

Inhibition of ERK/MAPK suppresses avian leukosis virus subgroup A and B replication.

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway contributes to subgroup J avian leukosis virus (ALV-J) replication and tumorigenicity. However, a role for ERK/MAPK signaling in ALV-A and ALV-B replication is unknown. In this study we successfully constructed and recovered a recombinant form of ALV-A strain GD13-1 which showed similarities in growth to the parental wild type virus in vitro. ALV subgroups J, A or B all triggered ERK2 activation in primary CEF cells. ERK/MAPK inhibition markedly suppressed ALV-A and ALV-B replication as shown by extremely low levels of viral transcription and virus protein production. This finding provides evidence that ERK/MAPK signaling responses play important roles in ALV replication and may represent novel drug targets for therapeutic intervention strategies.

Characterization and biological activity of Taishan Pinus massoniana pollen polysaccharide in vitro.

Taishan Pinus massoniana pollen polysaccharide (TPPPS) improves cellular and humoral immune responses of animals and is a novel potential immunomodulator. However, the components of TPPPS have not been recognized. To investigate the composition of TPPPS, crude polysaccharide was obtained from Taishan P. massoniana pollen through water extraction and ethanol precipitation. Three homogeneous polysaccharide fractions (TPPPS1, TPPPS2, and TPPPS3) were purified from TPPPS by DEAE-cellulose column chromatography. The average molecular weights of the three polysaccharides were 56, 25, and 128 kDa, respectively. Results of high-performance liquid chromatography (HPLC) showed that TPPPS comprised mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The biological activity assays showed that TPPPS2 and TPPPS3 significantly promoted spleen lymphocyte proliferation, and that TPPPS3 showed better effect than TPPPS2. TPPPS3 enhanced the secretion of cytokine IL-2 and TNF, whereas TPPPS2 mainly elevated IL-2 secretion. By contrast, TPPPS1 exhibited other effects, and it induced the highest amount of NO production, thereby indicating that TPPPS1 had the best antioxidant activity. TPPPS3 at 50 µg/mL significantly inhibited the proliferation of subgroup B Avian Leukosis virus (ALV-B) through virus adsorption interference in vitro. Results indicated that TPPPS comprised three main components, among which, TPPPS1 mainly showed antioxidant effects, whereas TPPPS2 and TPPPS3 played key roles in immunomodulation, especially TPPPS3. Further studies on the use of a reasonable proportion of TPPPS1-3 may facilitate the development of an effective immunomodulator.

Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design.

Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs.

BACKGROUND: Avian leukosis virus (ALV) is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi) is considered an effective antiviral strategy. RESULTS: In this study, the avian leukosis virus subgroup J (ALV-J) proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. The screened target (i.e., the gag genes) was shown to effectively suppress the replication of ALV-J by 19.0-77.3%. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes). Multi-target miRNAs were shown to play a synergistic role in the inhibition of ALV-J replication, with an inhibition efficiency of viral replication ranging from 85.0-91.2%. CONCLUSION: The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations.

Inhibition of Marek's disease virus replication by retroviral vector-based RNA interference.

RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short-hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In this study, similar RNAi vectors are shown to inhibit replication of the avian herpesvirus, Marek's disease virus (MDV, also known as gallid herpesvirus type 2), and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. We also describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and employ this vector with multiple targets within the MDV gB gene or within both the gB and ICP4 genes. The use of targets within multiple genes potentially can provide a larger antiviral effect and/or make it more difficult for viral escape mutations to evolve.

Expanded tropism and altered activation of a retroviral glycoprotein resistant to an entry inhibitor peptide.

The envelope of class I viruses can be a target for potent viral inhibitors, such as the human immunodeficiency virus type 1 (HIV-1) inhibitor enfuvirtide, which are derived from the C-terminal heptad repeat (HR2) of the transmembrane (TM) subunit. Resistance to an HR2-based peptide inhibitor of a model retrovirus, subgroup A of the Avian Sarcoma and Leukosis Virus genus (ASLV-A), was studied by examining mutants derived by viral passage in the presence of inhibitor. Variants with reduced sensitivity to inhibitor were readily selected in vitro. Sensitivity determinants were identified for 13 different isolates, all of which mapped to the TM subunit. These determinants were identified in two regions: (i) the N-terminal heptad repeat (HR1) and (ii) the N-terminal segment of TM, between the subunit cleavage site and the fusion peptide. The latter class of mutants identified a region outside of the predicted HR2-binding site that can significantly alter sensitivity to inhibitor. A subset of the HR1 mutants displayed the unanticipated ability to infect nonavian cells. This expanded tropism was associated with increased efficiency of envelope triggering by soluble receptor at low temperatures, as measured by protease sensitivity of the surface subunit (SU) of envelope. In addition, expanded tropism was linked for the most readily triggered mutants with increased sensitivity to neutralization by SU-specific antiserum. These observations depict a class of HR2 peptide-selected mutations with a reduced activation threshold, thereby allowing the utilization of alternative receptors for viral entry.

Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells.

Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate.

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.