Molecular dynamics simulations and conductance studies of the interaction of VP1 N-terminus from Polio virus and gp41 fusion peptide from HIV-1 with lipid membranes

The icosahedral Polio virus capsid consists of 60 copies of each of the coat proteins VP1, VP2, VP3 and myristolyated VP4 (myrVP4). Catalyzed by the host cell receptor the Polio virus enters the host cell via externalization of myrVP4 and the N terminal part of VP1. There are several assumptions about the individual role of both of the proteins in the mechanism of membrane attachment and genome injection. We use the first 32 N terminal amino acids of VP1 and applied molecular dynamics simulations to assess its mechanism of function when attached and inserted into hydrated lipid membranes (POPC). Helical models are placed in various positions in regard to the lipid membrane to start with. As a comparison, the first 33 amino acids of the fusion peptide of gp41 of HIV-1 are simulated under identical conditions. Computational data support the idea that VP1 is not penetrating into the membrane to form a pore; it rather lays on the membrane surface and only perturbs the membrane. Furthermore, this idea is strengthened by channel recordings of both peptides showing irregular openings.     

人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。     

Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.     

Membrane association of VP22, a herpes simplex virus type 1 tegument protein

Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U(L)49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.     

Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.     

Alpha4beta1 integrin acts as a cell receptor for murine polyomavirus at the postattachment level

The initial interaction of murine polyomavirus (Py) with host cells occurs through direct binding of the major capsid protein VP1 with cell membrane molecules containing terminal sialic acids; however, these Py receptor molecules have not yet been identified. Analysis of the capsid protein primary sequences of all murine strains revealed the presence of integrin ligand motifs in the DE and EF loops of VP1 (LDV and DLXXL, respectively) and at the N terminus of VP2 (DGE). We show that infectivity of the Py A2 strain in mouse Swiss 3T3 fibroblasts is significantly reduced only in the presence of natural integrin ligands carrying an LDV motif or antibodies directed against the alpha4 and beta1 integrin subunits. Furthermore, we demonstrate that expression of the alpha4 subunit in the alpha4-deficient BALB/c 3T3 cells increases viral infectivity. Addition of alpha4 function-blocking antibodies, prior to or after virus adsorption, blocks this increased infectivity without affecting virus binding to cells. Taken together, these data indicate that expression of alpha4 integrin enhances permissivity to Py, probably by acting as one of the postattachment receptors.     

The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection.

The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75 degrees C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.     

Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein

The rotavirus spike protein VP4 mediates attachment to host cells and subsequent membrane penetration. The VP8(*) domain of VP4 forms the spike tips and is proposed to recognize host-cell surface glycans. For sialidase-sensitive rotaviruses such as rhesus (RRV), this recognition involves terminal sialic acids. We show here that the RRV VP8(*)(64-224) protein competes with RRV infection of host cells, demonstrating its relevance to infection. In addition, we observe that the amino acids revealed by X-ray crystallography to be in direct contact with the bound sialic acid derivative methyl alpha-D-N-acetylneuraminide, and that are highly conserved amongst sialidase-sensitive rotaviruses, are residues that are also important in interactions with host-cell carbohydrates. Residues Arg101 and Ser190 of the RRV VP8(*) carbohydrate-binding site were mutated to assess their importance for binding to the sialic acid derivative and their competition with RRV infection of host cells. The crystallographic structure of the Arg(101)Ala mutant crystallized in the presence of the sialic acid derivative was determined at 295 K to a resolution of 1.9 A. Our multidisciplinary study using X-ray crystallography, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and competitive virus infectivity assays to investigate RRV wild-type and mutant VP8(*) proteins has provided the first evidence that the carbohydrate-binding cavity in RRV VP8(*) is used for host-cell recognition, and this interaction is not only with the sialic acid portion but also with other parts of the glycan structure.     

Ebola virus matrix protein VP40 uses the COPII transport system for its intracellular transport

The Ebola virus matrix protein VP40 plays an important role in virion formation and viral egress from cells. However, the host cell proteins and mechanisms responsible for intracellular transport of VP40 prior to its contribution to virion formation remain to be elucidated. Therefore we used coimmunoprecipitation and mass spectrometric analyses to identify host proteins interacting with VP40. We found that Sec24C, a component of the host COPII vesicular transport system, interacts specifically with VP40 via VP40 amino acids 303 to 307. Coimmunoprecipitation and dominant-negative mutant studies indicated that the COPII transport system plays a critical role in VP40 intracellular transport to the plasma membrane. Marburg virus VP40 was also shown to use the COPII transport system for intracellular transport. These findings identify a conserved intersection between a host pathway and filovirus replication, an intersection that can be targeted in the development of new antiviral drugs.     

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.     

Modeling a sialic acid binding pocket in the external loops of JC virus VP1

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.     

Asia1型口蹄疫病毒分离株结构蛋白基因的克隆与表达

口蹄疫病毒结构蛋白氨基酸的变化是病毒抗原性变异的分子基础,大部分抗原表位位于主要的免疫原蛋白VP1上,部分非线性抗原表位位于VP2和VP3上。本研究首次成功测定了 Asia1 型口蹄疫病毒(YNBS/58)四种结构蛋白基因( p1 区)的核苷酸序列,全长 2199 个碱基,编码 733 个氨基酸,该基因与 Ind63/72、Pka3/54、Israel、China/99、C1/Germany、A22、ZIM7/83/2 毒株的 p1 基因核苷酸序列同源性分别为 88. 4%、86. 0%、89. 3%、68.6%、67.6%、66.8%、50.3%,推导的氨基酸序列同源性分别为 94.1%、93.2%、95.1%、79.9%、77.0%、76.5%、58.1%;将YNBS/58株与 Ind63/72、Pka3/54、Israel株的 vp1、vp2、vp3、vp4 基因和编码蛋白分别进行同源性比较,发现VP1的序列变异最大,VP2、VP3、VP4次之,且VP1的氨基酸变异主要集中在 42-50 位和 137-156 位。实现了YNBS/58株结构蛋白基因在大肠杆菌中的高效表达,其表达的融合蛋白以包涵体形式存在,分子量约为88kDa,占菌体总蛋白的16%左右,并利用镍柱对目的蛋白进行了纯化,纯度达 90%以上,本实验为进一步研究 A sia1型口蹄疫病毒的分子流行病学、p1基因及其编码蛋白的生物学功能奠定了基础。     

不同宿主H1N1病毒血凝素蛋白(HA)受体结合位点的变异特征

2009年全球性爆发的H1N1病毒已经导致213个国家和地区受到感染, 有16 226人死亡。病毒与宿主细胞表面受体的结合是病毒感染不可缺少的第一步, 从而导致病毒膜与宿主细胞膜的融合。血凝素(Hemagglutinin, HA)就是介导这种受体结合与膜融合的病毒蛋白, 受体结合位点(Receptor binding sites, RBSs)位于HA蛋白三聚体中每个单体的球形头部, 主要由190位螺旋(190~198aa)、130位环(135~138aa)和220位环(221~228)3个二级结构域组成。文章收集了1918~2009年间1 221株H1N1病毒株的HA1序列(长度为327个氨基酸残基), 通过序列比对、各位点氨基酸残基的熵值以及3D结构模拟等生物信息学研究。结果显示不同宿主的不同病毒RBSs具有不同的熵值, 而且不同宿主的病毒HA1其RBSs具有不同的优势序列。3D结构模拟也显示了H1N1不同HA1之间在190位螺旋构象上的细微差异。该研究揭示了不同HA1上RBSs的一些新的特征, 为进一步探讨病毒感染的机理提供了新的信息     

A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus.

In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.     

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.     

Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence

Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.     

Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix-helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys-Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1-E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.     

Silencing the morphogenesis of rotavirus

The morphogenesis of rotaviruses follows a unique pathway in which immature double-layered particles (DLPs) assembled in the cytoplasm bud across the membrane of the endoplasmic reticulum (ER), acquiring during this process a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (TLPs). In this work, we have characterized the role of NSP4 and VP7 in rotavirus morphogenesis by silencing the expression of both glycoproteins through RNA interference. Silencing the expression of either NSP4 or VP7 reduced the yield of viral progeny by 75 to 80%, although the underlying mechanism of this reduction was different in each case. Blocking the synthesis of NSP4 affected the intracellular accumulation and the cellular distribution of several viral proteins, and little or no virus particles (neither DLPs nor TLPs) were assembled. VP7 silencing, in contrast, did not affect the expression or distribution of other viral proteins, but in its absence, enveloped particles accumulated within the lumen of the ER, and no mature infectious virus was produced. Altogether, these results indicate that during a viral infection, NSP4 serves as a receptor for DLPs on the ER membrane and drives the budding of these particles into the ER lumen, while VP7 is required for removing the lipid envelope during the final step of virus morphogenesis.     

Ebola virus VP40-induced particle formation and association with the lipid bilayer

Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.