Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity

The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP(5)), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP(5)-M heterodimerization and have identified the Cys residues involved in the formation of the GP(5)-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP(5) ectodomain or removal of the single GP(5) N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP(5) ectodomain had been deleted and the original Cys-80-->Ser mutation had been maintained. Consequently, this revertant lacked the GP(5) N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP(5) deletion, we confirmed that Cys-34 of GP(5) and Cys-8 of M are essential for GP(5)-M heterodimerization, a key event in the assembly of the EAV envelope.     

The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.     

Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly

Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 ((120)QAKKRVL(126)) and BR2 ((140)PGKKRPV(146)) had minor effects on viral infectivity ( approximately 4- and approximately 10-fold, respectively), whereas BR3 ((166)PARKRLN(172)) and BR4 ((307)RPKRLN(312)) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.     

Disulfide bonds are required for Serratia marcescens nuclease activity.

The role of the two disulfide bonds found in the Serratia marcescens nuclease were tested by site directed mutagenesis and were found essential for nuclease activity, although slight residual activity remained. The requirement for disulfide bond formation may play a role in preventing the lethal action of nuclease while in the bacterial cytoplasm.     

Disulfide bonds and membrane topology of the vaccinia virus A17L envelope protein

The envelope protein encoded by the vaccinia virus A17L open reading frame is essential for virion assembly. Our mutagenesis studies indicated that cysteines 101 and 121 form an intramolecular disulfide bond and that cysteine 178 forms an intermolecular disulfide linking two A17L molecules. This arrangement of disulfide bonds has important implications for the topology of the A17L protein and supports a two-transmembrane model in which cysteines 101 and 121 are intraluminal and cysteine 178 is cytoplasmic. The structure of the A17L protein, however, was not dependent on these disulfide bonds, as a recombinant vaccinia virus with all three cysteine codons mutated to serines retained infectivity.     

The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity

The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.     

Disulfide bonds and the stability of globular proteins.

An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect.     

Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity

The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.     

Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.     

Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity

Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.     

Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.     

Disulfide bonds of herpes simplex virus type 2 glycoprotein gB.

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.     

Conserved cysteine residues in the human immunodeficiency virus type 1 transmembrane envelope protein are essential for precursor envelope cleavage.

The transmembrane (TM) protein of human immunodeficiency virus type 1 has been demonstrated to be involved in viral infectivity and syncytium formation. Two highly conserved cysteine residues in the extracellular region of the TM protein are shown to be essential for processing the 160-kDa envelope precursor into the active 120- and 41-kDa mature forms.     

Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.     

Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus

The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins.     

Retention of viral infectivity after extensive mutation of the highly conserved immunodominant domain of the feline immunodeficiency virus envelope.

In lentiviruses, including human immunodeficiency virus and feline immunodeficiency virus (FIV), the principal immunodominant domain (PID) of the transmembrane glycoprotein elicits a strong humoral response in infected hosts. The PID is marked by the presence of two cysteines that delimit a sequence, composed of five to seven amino acids in different lentiviruses, which is highly conserved among isolates of the same lentiviral species. While the conservation of the sequence suggests the presence of functional constraints, the conservation of the immunodominance among divergent lentiviruses raises the hypothesis of a selective advantage for the infecting virus conferred by the host humoral response against this domain. We and others have previously shown that an appropriate structure of the PID is required for the production of a functional envelope. In the present work, we analyzed virological functions and immune reactivity of the envelope after random mutagenesis of the PID of FIV. We obtained nine mutant envelopes which were correctly processed and retained fusogenic ability. Mutation of the two C-terminal residues of the PID sequence between the cysteines in a molecular clone of FIV abolished infectivity. In contrast, three molecular clones containing extensive mutations in the four N-terminal amino acids were infectious. However, the mutations affected PID reactivity with sera from infected cats. Our results suggest that functional constraints, although existent, are not sufficient to account for PID sequence conservation. Such conservation may also result from positive selection by anti-PID antibodies which enhance infection.     

Role of conserved gp41 cysteine residues in the processing of human immunodeficiency virus envelope precursor and viral infectivity.

All animal retroviruses whose nucleotide sequences have been determined contain two or three closely spaced cysteine residues in the extracellular domain of the env-encoded transmembrane protein. Using human immunodeficiency virus type 1 gp41 as a working model, the functional significance of these highly conserved cysteines was investigated. We report here that substituting the two conserved cysteine residues in this domain of gp41 with glycine residues resulted in the loss of viral infectivity, which could be attributed to severe impairment in the processing of gp160 precursor to gp120.