Inhibition Of Virus Production Research Articles

Synthesis and the biological evaluation of 2-benzenesulfonylalkyl-5-substituted-sulfanyl-[1,3,4]-oxadiazoles as potential anti-hepatitis B virus agents

Current treatments for chronic hepatitis B virus (HBV) infection include the use of interferon-α and of nucleoside analogs lamivudine, adefovir and entecavir. However, the use of interferon-α has many side effects while that of nucleosidic inhibitors can lead to the emergence of resistant viruses. Hence, new drugs for the treatment of HBV infection are still highly desired. Oxadiazoles have been observed to exhibit antiviral activities against RNA viruses. In this study, a facile synthesis of 2-benzenesulfonylalkyl-5-substituted-sulfanyl-[1,3,4]-oxadiazoles is reported. The compounds were then evaluated for their anti-HBV activity. 1-{2-[5-(1-Benzenesulfonyl-propyl)-[1,3,4]oxadiazol-2-yl-sulfanyl]-ethyl}-4-(2-methoxy-phenyl)-piperazine ( 1i) was able to inhibit the expression of the viral antigens, HBsAg and HBeAg in a concentration-dependent manner with no cytotoxic effects and without any effects on the expression of viral transcripts. Concentration- and time-dependent reductions in virion production were also observed. The inhibition of virion production was comparable to that of lamivudine and EC 50 values of 1.63 and 2.96 μM were obtained for compound 1i and lamivudine, respectively. Thus, in addition to the antiviral effects on RNA viruses, oxadiazoles also have anti-HBV activities.

Potent and Specific Inhibition of Retrovirus Production by Coexpression of Multiple siRNAs Directed Against Different Regions of Viral Genomes

siRNA-mediated RNA degradation has been demonstrated to act as an antiviral system in many species. Here we describe inhibition of retrovirus production by multiple siRNAs designed to target various regions of the viral genomes. Using murine leukemia virus (MuLV) as a model, we demonstrate that the virus production can be inhibited by 77% in siLTR2 (a siRNA targeting the U3 region of MuLV) expression vector transfected cells. Coexpression of siLTR2 with siPsi2 (a siRNA targeting the 3' Psi (packaging signal sequence) results in 93% suppression of the virus production, suggesting that an increased inhibition of the virus production can be achieved by coexpression of multiple siRNAs to target different regions of the viral RNA simultaneously. Our results also indicate that not all sequences of the viral RNA are equally accessible to siRNA. We show that U3 region of MuLV is more accessible to siRNA, whereas the packaging signal sequence, especially the region adjacent to 5'LTR, is less accessible to siRNA, partly as a result of the binding of Gag precursors. Furthermore, we demonstrate that coexpression of siLTR2 with siPsi2 in virus producer cells leads to 88% knockdown of viral titer, showing the benefit of coexpression of multiple siRNAs for potent suppression of virus production in the setting of an established infection. Moreover, we demonstrate that infection of MuLV in cells that stably coexpress siLTR2 with siPsi2 diminishes by 77%. Taken together, we establish that siRNA-mediated gene silencing can suppress multiple steps of the retrovirus life cycle, offering a potential for both treating virus-associated diseases and preventing viral infection.

802. Silencing of HIV-1 with RNA Interference: A Multiple shRNA Approach

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). The double-stranded RNA can be expressed in the cell as a short hairpin RNA (shRNA) with a stem of 21–23 base pairs. Previously, we have shown that stable expression of a shRNA targeting the Nef gene of HIV-1 results in strong inhibition of HIV-1 replication. However, the application of this single shRNA was not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presented a way out for the virus, as single nucleotide substitutions in the target region abolished the inhibition. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs (ter Brake and Berkhout 2005). Therefore, we screened 86 different shRNAs targeting highly conserved regions. We identified multiple shRNAs that act as potent inhibitors of virus replication (Figure 1). We show, for the first time, that the expression of three different shRNAs from a single lentiviral vector results in similar levels of inhibition per shRNA as compared to single shRNA vectors. Thus, their combined expression results in an additive effect on inhibition of virus production. Moreover, when we expressed multiple shRNAs in a cell line that is susceptible for HIV-1 replication, viral escape was prevented. These results confirm that RNAi has great potential as antiviral gene therapy approach and supports our efforts to develop this strategy for the treatment of HIV-1 infected individuals. Brake, O. ter., and Berkhout, B. (Journal of RNAi and Gene Silencing 2005, 1(2): 56–65). Figure 1 86 shRNA expression plasmids were co-transfected with the HIV-1 molecularLAI and virus production measured. Figure 2Position of effective shRNA targets in the HIV-1 genome.

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Although the effective use of highly active antiretroviral therapy results in the suppression of virus production in infected individuals, it does not eliminate the infection and low level virus production in cells harboring virus in sanctuary sites. Thus, the continued search for new antiretroviral agents with unique and different mechanisms of HIV inhibition remains critical, and compounds that can reduce the level of virus production from cells already infected with HIV, as opposed to preventing de novo infection, would be of great benefit. A mercaptobenzamide (MDH-1-38) and its prodrug (NS1040) are being developed as potential therapeutic compounds targeting the zinc finger of HIV nucleocapsid. In the presence of esterase enzymes, NS1040 is designed to be converted to MDH-1-38 which has antiviral activity. While we presume that NS1040 is rapidly converted to MDH-1-38 in all experiments, the two compounds were tested side-by-side to determine whether the presence of a prodrug affects the antiviral activity or mechanism of action. The two compounds were evaluated against a panel of HIV-1 clinical isolates in human PBMCs and monocyte-macrophages and yielded EC50 values ranging from 0.7 to 13 μM with no toxicity up to 100 μM. MDH-1-38 and NS1040 remained equally active in human PBMCs in the presence of added serum proteins as well as against HIV-1 isolates resistant to reverse transcriptase, integrase or protease inhibitors. Cell-based and biochemical mechanism of antiviral action assays demonstrated MDH-1-38 and NS1040 were virucidal at concentrations of 15 and 50 μM, respectively. Cell to cell transmission of HIV in multiple passages was significantly reduced in CEM-SS and human PBMCs by reducing progeny virus infectivity at compound concentrations greater than 2 μM. The combination of either MDH-1-38 or NS1040 with other FDA-approved HIV drugs yielded additive to synergistic antiviral interactions with no evidence of antiviral antagonism or synergistic toxicity. Serial dose escalation was used in attempts to select for HIV strains resistant to MDH-1-38 and NS1040. Virus at several passages failed to replicate in cells treated at increased compound concentrations, which is consistent with the proposed mechanism of action of the virus inactivating compounds. Through 14 passages, resistance to the compounds has not been achieved. Most HIV inhibitors with mechanism of antiviral action targeting a viral protein would have selected for a drug resistant virus within 14 passages. These studies indicate that these NCp7-targeted compounds represent new potent anti-HIV drug candidates which could be effectively used in combination with all approved anti-HIV drugs.

BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.

Design, synthesis, and evaluation of dioxane-based antiviral agents targeted against the Sindbis virus capsid protein

Dioxane-based antiviral agents targeted to the hydrophobic binding pocket of Sindbis virus capsid protein were designed by computer graphics molecular modeling and synthesized. Virus production using SIN-IRES-Luc and capsid assembly were monitored to evaluate antiviral activity. A compound with a three-carbon linker chain connecting two dioxane moieties inhibited virus production by 50% at a concentration of 40 μM, while ( R)-hydroxymethyldioxane inhibited virus production by 50% at a concentration of 1 μM. Both compounds were not cytotoxic in uninfected BHK cells at concentrations of 1 mM.

The cellular HIV-1 Rev cofactor hRIP is required for viral replication

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after reintroduction of hRIP protein. Taken together, our results indicate that hRIP is an essential cellular cofactor for Rev function and HIV-1 replication. Because hRIP is not required for cell viability, it may be an attractive target for the development of new antiviral strategies.

IgG from acutely infected cats blocks mucosal feline immunodeficiency virus infection

We have previously shown an absence of detectable systemic or local infection in cats exposed to an infectious (100 TCID 50) feline immunodeficiency virus (FIV) plasma inoculum via either the rectal or vaginal mucosa (Burkhard, M.J., Obert, L.A., O’Neil, L.L., Diehl, L.J., Hoover, E.A., 1997. Mucosal transmission of cell-associated and cell-free feline immunodeficiency virus. AIDS Res. Hum. Retroviruses 13, 347–355). In contrast, this same plasma inoculum was infectious via parenteral inoculation. Moreover an equivalent dose of cell-free tissue culture-origin virus inoculum infected 100% of cats by either the rectal or vaginal exposure route. To evaluate this phenomena, we used a tissue culture system to identify a heat-stable factor in the plasma of cats acutely (3 weeks) infected with FIV that blocked infection of naive peripheral blood mononuclear cells (PBMC) by either cell-free or cell-associated FIV in vitro. A single application of as little as a 1:200 dilution of either heparinized or Alsevier's anticoagulated plasma effectively inhibited production of FIV p26 in culture over a 21-day co-culture period. Depletion of antibody using a protein A column abrogated the inhibitory effect of FIV plasma against in vitro FIV infection. Co-inoculation of heat-inactivated plasma with 400 TCID 50 FIV-B-2542 cell-free supernatant virus onto the vaginal mucosa of two cats resulted in complete inhibition of infection in one cat and increased time to infection in the second. Thus, antibody found in the plasma of cats acutely infected with FIV blocks cell-associated and cell-free infection, inhibits virus production in previously infected cells, and reduces mucosal transmission efficiency in vivo. Extrapolation may help explain the relatively inefficient mucosal transmission of human immunodeficiency virus-1 (HIV) and other lentiviruses.

Current and Future Concepts in Hepatitis C Therapy

The goal of hepatitis C virus (HCV) therapy is permanent viral eradication. This requires the use of drug combinations with multiple modes of action. Steady-state HCV replication kinetics can be disrupted by drugs that inhibit virus production (antiviral molecules), inhibit de novo cell infection, and/or accelerate the clearance of infected cells. Pegylated interferon-alpha and ribavirin combine all of these mechanisms of action when used together, yet fail to clear HCV from a significant number of patients. New therapeutic approaches are needed. The next generation of anti-HCV therapeutic agents will fall into four main categories: new interferons and interferon inducers, alternatives to ribavirin, specific HCV inhibitors, and immune therapies. Ideally, these new treatments will increase the rate of sustained viral eradication and improve tolerability and acceptability. Drug combinations will be tailored to the individual patient, based on baseline parameters and viral kinetics during therapy.

Antiviral Effects of Human Immunodeficiency Virus Type 1-Specific Small Interfering RNAs against Targets Conserved in Select Neurotropic Viral Strains

RNA interference, a natural biological phenomenon mediated by small interfering RNAs (siRNAs), has been demonstrated in recent studies to be an effective strategy against human immunodeficiency virus type 1 (HIV-1). In the present study, we used 21-bp chemically synthesized siRNA duplexes whose sequences were derived from the gp41 gene, nef, tat, and rev regions of viral RNA. These sequences are conserved in select neurotropic strains of HIV-1 (JR-FL, JR-CSF, and YU-2). The designed siRNAs exerted a potent antiviral effect on these HIV-1 strains. The antiviral effect was mediated at the RNA level (as observed by the down-regulation of the HIV-1-specific spliced transcript generating a 1.2-kbp reverse transcription [RT]-PCR product) as well as viral assembly on the cell membrane. Spliced transcripts (apart from the most abundant transcript generating a 1.2-kbp RT-PCR product) arising from an unspliced precursor likely contributed, albeit to a lesser extent, to the antiviral effect. The resultant progeny viruses had infectivities similar to that of input virus. We therefore conclude that these siRNAs interfere with the processing of the unspliced transcripts for the gp41 gene, tat, rev, and nef, eventually affecting viral assembly and leading to the overall inhibition of viral production. Apart from using the gp41 gene as a target, the conservation of each of these targets in the above-mentioned viral strains, as well as several primary isolates, would enable these siRNAs to be used as potent antiviral tools for investigations with cells derived from the central nervous system in order to evaluate their therapeutic potential and assess their utility in inhibiting HIV-1 neuropathogenesis and neuroinvasion.

Relationship of HSP70 to Temperature‐Dependency of Influenza Viral Infection

AbstractFor Abstract see ChemInform Abstract in Full Text.

The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 gag protein is important for virion assembly and release.

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an alpha-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

Relationship of HSP70 to temperature-dependency of influenza viral infection

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus the export of a new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP), RNA polymerases, and matrix protein 1 (M1), synthesized in the cytoplasm are imported into the nucleus and form a viral ribonucleoprotein complex (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus. It was found unexpectedly that the production of influenza virus was suppressed in Madin-Darby canine kidney cells at 41 degrees C, although viral proteins were synthesized, because nuclear export of vRNP is blocked by the dissociation of M1 from vRNP. It was also suggested that a certain protein(s) synthesized only at 41 degrees C inhibited the association of M1 with vRNP. The potential of heat-shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37 degrees C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1.

Inhibition of virus production in JC virus-infected cells by postinfection RNA interference.

RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.

Heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex.

The influenza virus genome replicates and forms a viral ribonucleoprotein complex (vRNP) with nucleoprotein (NP) and RNA polymerases in the nuclei of host cells. vRNP is then exported into the cytoplasm for viral morphogenesis at the cell membrane. Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) work in the nuclear export of vRNP by associating with it. It was previously reported that influenza virus production was inhibited in Madin-Darby canine kidney (MDCK) cells cultured at 41 degrees C because nuclear export of vRNP was blocked by the dissociation of M1 from vRNP (A. Sakaguchi, E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim, Virology 306:244-253, 2003). Previous data also suggested that a certain protein(s) synthesized only at 41 degrees C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37 degrees C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41 degrees C, showing that the export of NS2 was also disturbed at 41 degrees C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37 degrees C, suggesting that HSP70 does not influence NS2.

A glucocorticoid response element in the LTR U3 region of Friend murine leukaemia virus variant FIS-2 enhances virus production in vitro and is a major determinant for sex differences in susceptibility to FIS-2 infection in vivo

The nucleotide sequence of the Friend murine leukaemia virus variant FIS-2 LTR has high identity with the closely related Friend murine leukaemia virus (F-MuLV) LTR, except for the deletion of one direct repeat, a few point mutations and the generation of a glucocorticoid response element (GRE) in the U3 region. The GRE can mediate gene induction by glucocorticoids, mineral corticoids, progesterone and androgens, and it has been shown that incorporation of a GRE(s) within the LTR can increase the transcriptional activity of retroviral enhancers. We have previously reported an increased early virus replication in male mice compared with female mice when infected with a virus containing the FIS-2 LTR and have proposed that the GRE might contribute to this sex difference. In the present study, we introduced a single point mutation in the GRE and performed comparative studies in NIH 3T3 cells and in young adult male and female NMRI mice. We found that significantly more virus was produced from NIH 3T3 cells infected with wt FIS-2 than from cells infected with the FIS-2 GRE mutant and that this difference was further augmented by glucocorticoids. The glucocorticoid antagonist RU486 inhibited virus production in a dose-dependent manner. The wt FIS-2 disseminated significantly faster than the FIS-2 GRE mutant in both male and female mice. There was no significant difference in the dissemination rate between male and female mice infected with the FIS-2 GRE mutant. Hence, the GRE in the FIS-2 LTR is one determinant of the significant sex difference in susceptibility to FIS-2 infection.

Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity.

We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double mutations at asparagine (VP0 aa 69) and serine (VP0 aa 70) resulted in no cleavage of VP0 and 100% inhibition of virus production. Mutation of the asparagine into threonine or phenylalanine resulted in a low amount of cleaved VP0 and infectious virus was found. After passage of this mutated virus VP0 cleavage became more efficient and the growth rate of the virus became similar to wild-type SVDV. The passaged virus had mutated at the asparagine site; the threonine had changed into an alanine and the phenylalanine into a cysteine. When the serine was mutated no maturation cleavage was observed and no infectious virus could be derived. All the mutations resulted in RNA encapsidation. We conclude that in the case of SVDV the cleavage site between VP2 and VP4 is essential for the formation of infectious virus which is comparable to poliovirus. The serine of the VP0 site was more important than the asparagine in this respect.

First phase hepatitis c viral kinetics in previous nonresponders patients

A large proportion of patients fails to respond to treatment for hepatitis C. Initiation of interferon therapy is associated with a rapid first phase decline in viremia, reflecting inhibition of viral production or release from infected cells. We characterized first phase viral kinetics in previous nonresponder patients and compared the antiviral efficacy of interferon in nonresponders to that observed in naive patients. Twenty nonresponders with genotype 1 infection were evaluated. Ten received a single 15 mcg dose of interferon alfacon-1 and ten were given a 30 mcg dose. Viral kinetic data from previously untreated historical control patients with genotype 1 infection who received 9 mcg (n = 12) or 15 mcg (n = 13) of interferon alfacon-1 provided a basis for comparison. Antiviral efficacy was evaluated by calculating the reduction in HCV RNA levels during the first 24 h after interferon administration (log effectiveness). Hepatitis C virus levels decreased exponentially in previous nonresponder patients. Non-responders treated with 30 mcg of interferon alfacon-1 exhibited a greater log drop than non-responders receiving 15 mcg (P = 0.01). The log effectiveness of 15 mcg of interferon alfacon-1 in nonresponders was similar to 9 mcg in naives and was significantly < 15 mcg (P = 0.04) in naïve patients. The 30 mcg dose provided similar log effectiveness in nonresponders compared with 15 mcg in naive patients and exceeded the log effectiveness of 9 mcg in previously untreated patients (P = 0.035). Nonresponders who had greater than a 50% decrease in HCV RNA level from baseline at the end of previous treatment had a larger reduction in viral load at 24 h compared with those who had not achieved that level of response with prior therapy (P = 0.04). In conclusion, the log effectiveness of interferon was lower in nonresponders compared with treatment naive patients. The difference in antiviral effectiveness in previous nonresponders was overcome by higher interferon doses.

SiRNA-directed inhibition of HIV-1 infection.

RNA interference silences gene expression through short interfering 21 23-mer double-strand RNA segments that guide mRNA degradation in a sequence-specific fashion. Here we report that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein. siRNAs effectively inhibit pre- and/or post-integration infection events in the HIV-1 life cycle. Thus, siRNAs may have potential for therapeutic intervention in HIV-1 and other viral infections.

Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function.

Efficient budding of HIV-1 from the plasma membrane of infected cells requires the function of a 6-kDa protein known as p6. A highly conserved Pro-Thr-Ala-Pro (PTAP) motif (the "late" or "L" domain), is critical for the virus-budding activity of p6. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N terminus a domain highly related to ubiquitin-conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle assembly and release. We observed that this domain (referred to as TSG-5') potently inhibits virus production. Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' depends on an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and indicate that TSG101 derivatives can act as potent and specific inhibitors of HIV-1 replication by blocking virus budding.