Infection of Tumour Cells by Fowl Plague Virus in the Presence of Actinomycin D

INFLUENZA virus is one of the few viruses in which replication is inhibited by the antimetabolite actinomycin D (AM-D)^1–4, which inhibits DNA-dependent RNA synthesis in mammalian cells⁵. It has been reported that the growth of fowl plague virus (FPV) in virus-transformed hamster cells is less sensitive to AM-D^6,7. We have examined the sensitivity of FPV to AM-D to see whether it is related to differences in the oncogenic properties of tumour cells. We found that in cells transformed by polyoma virus (PV) and also in cells transformed by methylcholanthrene, although no infectious virus was produced the cells synthesized viral haemagglutinin (HA). It was only the cell-associated HA, however, that was affected by AM-D and not that released by the cells.     

表达鸡新城疫病毒F、HN基因和传染性喉气管炎病毒gB基因的重组鸡痘病毒的构建及其特性研究

利用同源重组将新城疫病毒(NDV)的F和HN基因、传染性喉气管炎病毒(ILTV)的gB基因以及报告基因LacZ插入鸡痘病毒(FPV)的017株的复制非必需区,其中NDV的F、HN基因、ILTV的gB基因以及报告基因LacZ是在早晚期启动子LP2EP2的控制下,大肠杆菌报告基因LacZ在晚期启动子P11的控制下。经过10轮蓝斑纯化获得了包含了NDV的F和HN基因、ILTV的gB基因以及报告基因LacZ的重组鸡痘病毒,称为rFPV-F/HN/gB/LacZ。经PCR方法证明rFPV-F/HN/gB/LacZ基因组中含有NDV的F基因、HN基因和ILTVgB基因;间接免疫荧光试验和Western-blot试验表明NDV的F、HN蛋白和ILTVgB蛋白在rFPV-F/HN/gB/LacZ感染的CEF细胞中获得表达。与亲本毒相比,重组病毒在病毒的复制和致鸡胚成纤维细胞的病变方面无显著不同。这证明了在鸡痘病毒载体的一个复制非必需区可以同时插入多个禽类病原的多个外源基因,为制备多价基因工程疫苗奠定了基础。     

An acidic cluster of the cytoplasmic tail of the RD114 virus glycoprotein controls assembly of retroviral envelopes

Retroviral core proteins, Gag and envelope (Env) glycoproteins are expressed from distinct cellular areas and therefore need to encounter to assemble infectious particles. The intrinsic cell localisation properties of either viral component or their capacity to mutually interact determines the assembly of infectious particles. Here, we address how Env determinants and cellular sorting proteins allow the Env derived from gamma retroviruses, murine leukemia virus (MLV) and RD114, to travel to or from late endosomes (LE), which may represent the Env assembly site of retroviruses in some cells. The individual expression of MLV Env resulted in its accumulation in LE in contrast to RD114 Env that required the presence of gamma retroviral Gag proteins. To discriminate between intrinsic intracellular Env localisation and gamma retroviral Gag/Env interactions in influencing Env viral incorporation, we studied Env assembly on heterologous lentiviral particles on which they are passively recruited. We found that an acidic cluster present at the C-terminus of the RD114 Env cytoplasmic tail determines its sub-cellular localisation and retrograde transport. Mutation of this motif induced late endosomal concentration of the RD114 Env, correlating with increased viral incorporation and infectivity. Reciprocally, the reinforcement of a poorly functional acidic motif in the MLV Env resulted in a marked decrease of its late endosomal localisation, leading to weakly infectious lentiviral particles with low Env densities. Finally, through upregulation versus downregulation of its cellular expression, we show that phosphofurin acidic-cluster-sorting protein 1 (PACS-1) controls the function of the RD114 Env acidic cluster, assigning to this cellular effector a crucial role in modulation of Env assembly of some retroviruses.     

表达H5N1亚型禽流感病毒HA蛋白的重组鼠白血病病毒的特性

通过反转录 聚合酶链式反应 (RT PCR)扩增了H5N1亚型鹅源禽流感病毒 (AIV)完整的血凝素 (HA)基因并进行了克隆与鉴定。序列测定结果已经登陆GenBank ,登陆号为AY6 394 0 5。序列分析表明所扩增的HA基因开放性阅读框架 (ORF)由170 7个核苷酸组成 ,共编码 5 6 8个氨基酸 ,裂解位点的氨基酸组成为RKKR↓GLF ,含连续的碱性氨基酸 ,具有高致病性AIVHA基因裂解位点的特征。构建了含HA基因的真核表达载体pcDNA HA ,通过与鼠白血病病毒 (MuLV)假病毒构建体系的两种质粒pHIT6 0和pHIT111共转染人胚肾细胞 2 93T ,4 8h后收集假病毒上清 ,超离后通过Western blot证明HA蛋白能够在假病毒颗粒表面表达 ,表明HA能够整合到此病毒粒子表面。通过感染 2 93T、COS 7和NIH3T3三种不同的靶细胞 ,证实所构建的假病毒粒子具有感染性和泛嗜性。本研究成功构建了具有感染性的MuLV HA假病毒体系 ,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.     

Spontaneous heteromerization of gammaretrovirus envelope proteins: a possible novel mechanism of retrovirus restriction

The env gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral env genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.     

Mobilization of full-length Semliki Forest virus replicon by retrovirus particles

Conciliating biosafety with efficient gene transfer remains a constant concern in the development of retroviral vectors. Semliki Forest virus (SFV) replicons allow important retroviral vector production with interesting features. It is noteworthy that retroviruses have the ability to package Psi+ and, to some extent, Psi- cellular RNAs. Therefore, it was important to study the retroviral transfer of highly abundant SFV genomes expressing retroviral proteins. Here, we show that full-length SFV-vector replicons, with or without Psi, are efficiently packaged into retrovirus particles. Mechanistically, our data suggest that SFV packaging is the sum of its retroviral nucleocapsid-dependent recruitment together with a passive hijacking of membrane-anchored SFV replicon. A direct consequence of this phenomenon is the formation of particles harboring autonomous replicative abilities and contaminating vector preparations. Importantly, we confirm that retroviral SFV mobilization is not an exclusive feature of murine gamma retroviruses, since it is also observed using lentivectors.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

Regulation of Virus Release by the Macrophage-Tropic Human Immunodeficiency Virus Type 1 AD8 Isolate Is Redundant and Can Be Controlled by either Vpu or Env

The human immunodeficiency virus type 1 (HIV-1) Vpu and Env proteins are expressed from a bicistronic mRNA. To address the biological significance of the coordinated expression of vpu and env, we compared the relative effects on particle release of HIV-1 isolates containing an intact vpu gene or carrying point mutations in its initiation codon or internal deletions, respectively. We found that the primary AD8 isolate, which is unable to express vpu due to a mutation in its translation initiation codon, was able to replicate in primary macrophages and peripheral blood mononuclear cells with efficiency similar to that of an isogenic variant expressing Vpu. Interestingly, AD8 lacking a vpu initiation codon produced higher levels of Env protein than its Vpu-expressing isogenic variant. In contrast, disabling Vpu without removing the vpu initiation codon did not alter Env expression but significantly reduced virus production. AD8 Env when provided in trans was capable of enhancing release not only of AD8 particles but also of viruses of the T-cell-tropic NL4-3 isolate. We conclude that AD8 Env encodes a Vpu-like activity similar to that previously reported for HIV-2 Env proteins and is thus able to augment virus secretion. When expressed at elevated levels, i.e., following mutation of the vpu initiation codon, AD8 Env was able to compensate for the lack of Vpu and thereby ensure efficient virus release. Thus, the ability to regulate virus release is redundant in AD8 and can be controlled by either Vpu or Env. Since Vpu controls several independent functions, including CD4 degradation, our results suggest that some HIV-1 isolates may have evolved a mechanism to regulate Vpu activity without compromising their ability to efficiently replicate in the host cells.     

Site-specific S-Acylation of Influenza Virus Hemagglutinin: THE LOCATION OF THE ACYLATION SITE RELATIVE TO THE MEMBRANE BORDER IS THE DECISIVE FACTOR FOR ATTACHMENT OF STEARATE*

S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [³H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern.     

Late assembly motifs of human T-cell leukemia virus type 1 and their relative roles in particle release

Three late assembly domain consensus motifs, namely PTAP, PPPY, and LYPXL, have been identified in different retroviruses. They have been shown to interact with the cellular proteins TSG101, Nedd4, and AP2 or AIP, respectively. Human T-cell leukemia virus type 1 (HTLV-1) has a PPPY and a PTAP motif, separated by two amino acids, located at the end of MA, but only the PPPY motif is conserved in the deltaretrovirus group. Like other retroviral peptides carrying the late motif, MA is mono- or di-ubiquitinated. A mutational analysis showed that 90% of PPPY mutant particles were retained in the cell compared to 15% for the wild-type virus. Mutations of the PTAP motif resulted in a 20% decrease in particle release. In single-cycle infectivity assays, the infectious titers of late motif mutants correlated with the amounts of released virus, as determined by an enzyme-linked immunosorbent assay. We observed binding of MA to the WW domains of the Nedd4 family member WWP1 but not to the amino-terminal ubiquitin E2 variant domain of TSG101 in mammalian two-hybrid analyses. The binding to WWP1 was eliminated when the PPPY motif was mutated. However, MA showed binding to TSG101 in the yeast two-hybrid system that was dependent on an intact PTAP motif. A dominant-negative (DN) mutant of WWP1 could inhibit budding of the intact HTLV-1 virus. In contrast, DN TSG101 only affected the release of virus-like particles encoded by Gag expression plasmids. Electron and fluorescent microscopy showed that Gag accumulates in large patches in the membranes of cells expressing viruses with PPPY mutations. Very few tethered immature particles could be detected in these samples, suggesting that budding is impaired at an earlier step than in other retroviruses.     

Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase.

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.     

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78^Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.     

The GLN family of murine endogenous retroviruses contains an element competent for infectious viral particle formation

Several families of endogenous retroviruses (ERVs) have been identified in the mouse genome, in several instances by in silico searches, but for many of them it remains to be determined whether there are elements that can still encode functional retroviral particles. Here, we identify, within the GLN family of highly reiterated ERVs, one, and only one, copy that encodes retroviral particles prone to infection of mouse cells. We show that its envelope protein confers an ecotropic host range and recognizes a receptor different from mCAT1 and mSMIT1, the two previously identified receptors for other ecotropic mouse retroviruses. Electron microscopy disclosed viral particle assembly and budding at the cell membrane, as well as release of mature particles into the extracellular space. These particles are closely related to murine leukemia virus (MLV) particles, with which they have most probably been confused in the past. This study, therefore, identifies a new class of infectious mouse ERVs belonging to the family Gammaretroviridae, with one family member still functional today. This family is in addition to the two MLV and mouse mammary tumor virus families of active mouse ERVs with an extracellular life cycle.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.