The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.     

Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is one of four glycoproteins necessary and sufficient for HSV cellular entry. Recently, the crystal structures of HSV-1 gB and vesicular stomatitis virus glycoprotein G were determined. Surprisingly, the two proteins share remarkable structural homology. Both proteins are homotrimeric and center about a long alpha-helix, features reminiscent of class I fusion proteins, such as influenza virus hemagglutinin or paramyxovirus F. However, these structures revealed that G has internal fusion loops, similar to the fusion loops of the class II fusion proteins, and that these loops are structurally conserved in gB. To examine whether these putative fusion loops are important for gB function, we mutated potential membrane-interacting (hydrophobic) residues to charged amino acids. Of most interest were mutant gB proteins that were expressed on the cell surface and were recognized by monoclonal antibodies against conformational epitopes but lacked the ability to function in cell-cell fusion assays. We find that three of the five hydrophobic amino acids targeted in these loops, tryptophan 174, tyrosine 179, and alanine 261, are integral in the function of gB. Our data suggest that they are part of an important functional domain. We hypothesize that two loops in domain 1 of HSV gB function as fusion loops. Our data are further evidence that gB is a viral fusogen and suggest clues as to how gB may function.     

Mutational analysis of the zippering reaction during flavivirus membrane fusion

The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra- and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.     

Structural and functional properties of an unusual internal fusion peptide in a nonenveloped virus membrane fusion protein

The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.     

Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.     

Critical role for the cysteines flanking the internal fusion peptide of avian sarcoma/leukosis virus envelope glycoprotein

The transmembrane subunit (TM) of the envelope glycoprotein (Env) of the oncovirus avian sarcoma/leukosis virus (ASLV) contains an internal fusion peptide flanked by two cysteines (C9 and C45). These cysteines, as well as an analogous pair in the Ebola virus GP glycoprotein, are predicted to be joined by a disulfide bond. To examine the importance of these cysteines, we mutated C9 and C45 in the ASLV subtype A Env (EnvA), individually and together, to serine. All of the mutant EnvAs formed trimers that were composed of the proteolytically processed surface (SU) and TM subunits. All mutant EnvAs were incorporated into murine leukemia virus pseudotyped virions and bound receptor with wild-type affinity. Nonetheless, all mutant EnvAs were significantly impaired ( approximately 1,000-fold) in their ability to support infectivity. They were also significantly impaired in their ability to mediate cell-cell fusion. Our data are consistent with a model in which the internal fusion peptide of ASLV-A EnvA exists as a loop that is stabilized by a disulfide bond at its base and in which this stabilized loop serves an important function during virus-cell fusion. The fusion peptide of the Ebola virus GP glycoprotein may conform to a similar structure.     

The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization.

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.     

The pH sensor for flavivirus membrane fusion

Viruses that infect cells by uptake through endosomes have generally evolved to "sense" the local pH as part of the mechanism by which they penetrate into the cytosol. Even for the very well studied fusion proteins of enveloped viruses, identification of the specific pH sensor has been a challenge, one that has now been met successfully, for flaviviruses, by Fritz et al. (Fritz, R., K. Stiasny, and F.X. Heinz. 2008. J. Cell Biol. 183:353-361) in this issue. Thorough mutational analysis of conserved histidine residues in the envelope protein of tick-borne encephalitis virus led Fritz et al. (2008) to identify a histidine at a key domain interface as the critical pH sensor; its protonation triggers the large-scale conformational rearrangement that induces fusion of viral and endosomal membranes.     

A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.     

Mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by R peptide

During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.     

Mutational analysis of the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.     

Identification of the lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae

The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.     

Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization

The Flavivirus genus of the Flaviviridae family includes 70 enveloped single-stranded-RNA positive-sense viruses transmitted by arthropods. Among these viruses, there are a relevant number of human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), Langat virus (LGTV) and Omsk hemorrhagic fever (OHFV). The flavivirus envelope (E) protein is a dominant antigen inducing immunologic responses in infected hosts and eliciting virus-neutralizing antibodies. The domain III (DIII) of E protein contains a panel of important epitopes that are recognized by virus-neutralizing monoclonal antibodies. Peptides of the DIII have been used with promising results as antigens for flavivirus serologic diagnosis and as targets for immunization against these viruses. We review here some important aspects of the molecular structure of the DIII as well as its use as antigens for serologic diagnosis and immunization in animal models.     

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.     

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

A model of the tick-borne encephalitis virus envelope protein E is presented that contains information on the structural organization of this flavivirus protein and correlates epitopes and antigenic domains to defined sequence elements. It thus reveals details of the structural and functional characteristics of the corresponding protein domains. The localization of three antigenic domains (composed of 16 distinct epitopes) within the primary structure was performed by (i) amino-terminal sequencing of three immunoreactive fragments of protein E and (ii) sequencing the protein E-coding regions of seven antigenic variants of tick-borne encephalitis virus that had been selected in the presence of neutralizing monoclonal antibodies directed against the E protein. Further information about variable and conserved regions was obtained by a comparative computer analysis of flavivirus E protein amino acid sequences. The search for potential T-cell determinants revealed at least one sequence compatible with an amphipathic alpha-helix which is conserved in all flaviviruses sequenced so far. By combining these data with those on the location of disulfide bridges (T. Nowak and G. Wengler, Virology 156:127-137, 1987) and the structural characteristics of epitopes, such as dependency on conformation or on intact disulfide bridges or both, a model was established that goes beyond the location of epitopes in the primary sequence and reveals features of the folding of the polypeptide chain, including the generation of discontinuous protein domains.     

The pH dependence of flavivirus envelope protein structure: insights from molecular dynamics simulations

The flavivirus membrane fusion is triggered by the acid pH of the endosomes after virus endocytosis. The proposed mechanism involves changes in the protonation state of conserved histidine residues of the E protein present in the viral surface that undergoes a series of structural rearrangements that result in the fusion between the endosome and viral bilayers. We studied the pH dependence of E protein rearrangements of dengue virus type 2, used as a model, in the pH range experimented by the virus along the fusion process. We employed a low computational cost scheme to explore the behavior of the E protein by molecular dynamics (MD) simulations of complete systems that include the protein, the solvent, and ions. The procedure alternates cyclically the update of the ionization states of the protein residues with common MD steps applied to the new ionization configuration. Important pH-dependent protein structure rearrangements consistent with the changes of the protonation states of conserved histidine residues were observed. The involvement of other conserved residues in the flavivirus in the rearrangements was also identified. The results show interesting correlations with a proposed model for the fusion mechanism, as well as the experimentally identified key residues, contributing to a better understanding of the structural changes in protein E that lead to the fusion process.     

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.     

Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

The central proline of an internal viral fusion peptide serves two important roles

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope protein (Env) is internal, near the N terminus of its transmembrane (TM) subunit. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV fusion peptide and immediate surrounding sequences indicate a region of order (beta-sheet), a tight reverse turn containing the proline, and a second region of order (alpha-helix). Similar motifs (order, turn or loop, order) are predicted for other internal fusion peptides. In this study, we made and analyzed 12 Env proteins with substitutions for the central proline of the fusion peptide. Env proteins were expressed in 293T cells and in murine leukemia virus pseudotyped virions. We found the following. (i) All mutant Envs form trimers, but when the bulky hydrophobic residues phenylalanine or leucine are substituted for proline, trimerization is weakened. (ii) Surprisingly, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse turn. (iii) Nonetheless, proteolytically processed forms of all Envs are preferentially incorporated into pseudotyped virions. (iv) All Envs bind receptor with affinity greater than or equal to wild-type affinity. (v) Residues that support high infectivity cluster with proline at intermediate hydrophobicity. Infectivity is not supported by mutant Envs in which charged residues are substituted for proline, nor is it supported by the trimerization-defective phenylalanine and leucine mutants. Our findings suggest that the central proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.     

Immunomodulation by peptide analogs of retroviral envelope protein

The mechanism by which retroviral proteins exert their immunosuppressive influence has remained enigmatic. Early studies have demonstrated that retroviral infection suppresses cellular and humoral immune responses. A hydrophilic 26 amino acid region of the otherwise hydrophobic transmembrane envelope protein of murine and feline leukemia viruses, p15E, is conserved among the transmembrane envelope proteins of numerous animal retroviruses (e.g. murine, feline, bovine and simian) as well as in human T-cell leukemia virus, and to a lesser extent, in human immunodeficiency virus (HIV). We evaluated the immunomodulatory properties of various synthetic retroviral envelope peptides synthesized as overlapping fragments to this conserved sequence. We report that two small peptides inhibit human mixed lymphocyte reaction (MLR), interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) production. These peptides did not affect human natural killer (NK) cell cytotoxicity in vitro, and nitric oxide (NO) production in mouse macrophage cells, RAW264.7. Our observations suggests immunomodulatory potential of two retroviral peptide analogs.