Freeze-Drying of Foot-and-Mouth Disease Virus and Storage Stability of the Infectivity of Dried Virus at 4 C

Foot-and-mouth disease virus, type A, strain 119, propagated in cultures of calf kidney cells and in the tongue epithelium of cattle was used. The process of freeze-drying was conducted in two cycles on unit volumes of 4 ml in Pyrex ampoules, averaging 150 ampoules per run, and was studied separately from the problems of storage. Ampoules containing freeze-dried virus were flame-sealed for either immediate study or storage at 4 C for later reference. Tissue-culture virus dried with various additives had a mean processing loss of 0.8 log LD₅₀ per ml for six different preparations. Virus freeze-dried in tissue suspension had a mean loss of 0.8 log LD₅₀ per ml for three different preparations. A second set of preparations was processed and specifically studied for storage quality at 4 C. The virus in 14 freeze-dried tissue-culture preparations had a mean loss of 0.75 log LD₅₀ per ml while stored at 4 C for 1 year. Virus in four freeze-dried tissue suspensions had a mean loss of 0.05 log LD₅₀ per ml held at 4 C for 1 year. None of the specific additives used for conservation of the virus during the freeze-drying process or during storage at 4 C contributed significantly to the stability of the virus preparations over and above that observed with the normal growth medium of the tissue culture or the ordinary diluents used in making suspensions of tissue virus.     

乙肝病毒核衣壳启动子内三链DNA的形成

用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ（ＣＰ１）与１２９ｂｐ的乙肝病毒（ＨＢＶ）核衣壳启动子（Ｃｐ）片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性．在克隆有ＨＢＶ基因组的质粒ｐＣＰ１０的酶切产物中,ＣＰ１仅与含Ｃｐ的１２９ｂｐ片段结合．在２０ｍｍｏｌ／ＬＭｇ２＋溶液中其解离常数（Ｋｄ）为１．４×１０－７ｍｏｌ／Ｌ．不同离子稳定三链ＤＮＡ的效果依次为ｓｐ４＋（精胺）＞Ｍｇ２＋＞Ｚｎ２＋＞Ｎａ＋＞Ｋ＋,离子之间存在相互竞争作用．比ＣＰ１多一误配碱基的脱氧寡核苷酸Ｇ２ＴＧ２ＴＧＴＧ３ＴＧ２ＴＧ２ＴＧ２Ｔ（ＣＰ２）在２０ｍｍｏｌ／ＬＭｇ２＋溶液中与Ｃｐ结合的Ｋｄ值约为ＣＰ１的１／７,而在６０ｍｍｏｌ／ＬＫ＋或５ｍｍｏｌ／ＬＺｎ２＋溶液中检测不到它与Ｃｐ的结合,这进一步显示了三链ＤＮＡ形成的特异性．细胞的生理离子浓度被认为是：Ｓｐ４＋１ｍｍｏｌ／Ｌ,Ｍｇ２＋１０ｍｍｏｌ／Ｌ,Ｋ＋１４０ｍｍｏｌ／Ｌ,因此,ＣＰ１在细胞内将能特异地与Ｃｐ结合并具有较好的稳定性．     

Within-Host Spatiotemporal Dynamics of Plant Virus Infection at the Cellular Level

A multicellular organism is not a monolayer of cells in a flask; it is a complex, spatially structured environment, offering both challenges and opportunities for viruses to thrive. Whereas virus infection dynamics at the host and within-cell levels have been documented, the intermediate between-cell level remains poorly understood. Here, we used flow cytometry to measure the infection status of thousands of individual cells in virus-infected plants. This approach allowed us to determine accurately the number of cells infected by two virus variants in the same host, over space and time as the virus colonizes the host. We found a low overall frequency of cellular infection (<0.3), and few cells were coinfected by both virus variants (<0.1). We then estimated the cellular contagion rate (R), the number of secondary infections per infected cell per day. R ranged from 2.43 to values not significantly different from zero, and generally decreased over time. Estimates of the cellular multiplicity of infection (MOI), the number of virions infecting a cell, were low (<1.5). Variance of virus-genotype frequencies increased strongly from leaf to cell levels, in agreement with a low MOI. Finally, there were leaf-dependent differences in the ease with which a leaf could be colonized, and the number of virions effectively colonizing a leaf. The modeling of infection patterns suggests that the aggregation of virus-infected cells plays a key role in limiting spread; matching the observation that cell-to-cell movement of plant viruses can result in patches of infection. Our results show that virus expansion at the between-cell level is restricted, probably due to the host environment and virus infection itself.     

Herpes-Type Virus of the Frog Renal Adenocarcinoma: I. Virus Development in Tumor Transplants Maintained at Low Temperature

Development of the herpes-type virus of the frog kidney tumor was investigated by electron microscopy and high-resolution autoradiography in eyechamber transplants of tumor maintained at 7.5 C for up to 27 weeks. Virus particles were first detected at 10 weeks in nuclei containing aggregates of dense granular material. The initial incorporation of a pulse of (3)H-thymidine into these aggregates indicated that they contained newly synthesized viral deoxyribonucleic acid. Capsids enclosing doubleshelled cores were labeled with (3)H-thymidine before capsids with dense cores, and intermediate core forms were observed, suggesting that the double-shelled core transforms into the dense core. Particles with dense cores were observed while being enveloped by budding through the inner membrane of the nuclear envelope, and subsequently while being unenveloped in passing through the outer membrane into the cytoplasm. Virus particles within the cytoplasm acquired fibrillar coats and budded into vesicles, from which they were released, in enveloped form, at the cell surface. Tubular forms and particles considerably smaller than virus particles were regularly encountered in infected nuclei, and the relationship of these forms to virus replication is discussed.     

Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol_DV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol_DV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn²⁺ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol_DV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg²⁺. In the presence of Mn²⁺, NS5Pol_DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.     

Rescue of Simian Virus 40 from Cell Lines Transformed at High and at Low Input Multiplicities by Unirradiated or Ultraviolet-irradiated Virus

The relation between simian virus 40 (SV40) input multiplicity during transformation of primary mouse kidney cultures and the subsequent rescue of SV40 from clonal lines of transformed cells has been studied. Primary mouse kidney cultures were transformed with unirradiated SV40 at input multiplicities varying from 0.06 to 200 plaque-forming units (PFU) /cell or with SV40 irradiated with ultraviolet (UV) light to a survival of 0.04 to 0.01. All of the transformed lines contained the intranuclear SV40 T antigen, but cell-free extracts prepared from the transformed cell lines failed to yield infectious virus when assayed on monkey kidney cell (CV-1) monolayers. After fusion with susceptible CV-1 cells induced by UV-inactivated Sendai, all of the lines transformed by unirradiated virus yielded infectious SV40. The frequency of induction and the incidence of successful trials did not depend on the multiplicity of infection. “Good” yielders were obtained from mouse kidney cells transformed at the low input multiplicity of 0.06 PFU /cell. In contrast, only 4 of 12 clonal lines transformed at moderately low input multiplicity, and none of the lines transformed at very low input multiplicity with UV-irradiated virus yielded infectious SV40. The four positive lines have been classified as “poor” or “rare” yielders.     

Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.     

RNA Sequence Features Are at the Core of Influenza A Virus Genome Packaging

The influenza A virus (IAV), a respiratory pathogen for humans, poses serious medical and economic challenges to global healthcare systems. The IAV genome, consisting of eight single-stranded viral RNA segments, is incorporated into virions by a complex process known as genome packaging. Specific RNA sequences within the viral RNA segments serve as signals that are necessary for genome packaging. Although efficient packaging is a prerequisite for viral infectivity, many of the mechanistic details about this process are still missing. In this review, we discuss the recent advances toward the understanding of IAV genome packaging and focus on the RNA features that play a role in this process.     

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.     

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.     

Limited Interference at the Early Stage of Infection between Two Recombinant Novirhabdoviruses: Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus

The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-ΔNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFP_max reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFP_max could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFP_max and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins.Viral hemorrhagic septicemia virus (VHSV) is a member of the Novirhabdovirus genus in the Rhabdoviridae family. VHSV is considered by many countries and international organizations to be one of the most important viral pathogens of finfish (38). During recent years, VHSV has been isolated from at least 50 different species from marine and freshwater fish and is present throughout the northern hemisphere (45). The transmission of the virus from fish to fish occurs directly through the water or by contact between infected and healthy individuals. VHSV is thought to enter the body through the gills or possibly through wounds on the skin. However, we recently showed that fins may represent the main portal of entry for the novirhabdoviruses (25). The virus usually causes severe hemorrhages in the skin, muscles, eyes, kidney, and liver, with mortality rates as high as 90%. As for all members of the Rhabdoviridae family, the VHSV genome consists of a negative-sense single-stranded RNA molecule of about 11 kb encoding five structural proteins: N, the nucleoprotein; P, a polymerase-associated protein; M, the matrix protein; G, the unique viral surface glycoprotein; and L, the large RNA-dependent RNA polymerase. In addition, like the other members of the Novirhabdovirus genus, such as infectious hematopoietic necrosis virus (IHNV), the VHSV genome encodes a small nonstructural NV protein, which has been shown to be dispensable for IHNV replication in cell culture and is involved in virus-induced pathogenicity in rainbow trout (8, 50).The sequence analysis of the glycoprotein (G) and nucleoprotein (N) genes of VHSV has shown that VHSV isolates can be divided into four genotypes that generally correlate with geographic location rather than the host species (4, 19, 47, 49). Isolates belonging to VHSV genotypes I, II, and III are present in continental Europe, the north Atlantic Ocean, the Baltic Sea, the North Sea, and waters around Scotland. Genotype IV consists of isolates from the marine environment in North America. Recently, viral hemorrhagic septicemia has become an emerging disease of freshwater fish in the Great Lakes region of North America (2, 54). Thus, it is quite obvious that VHSV is becoming a worldwide and very-broad-host-range fish virus and that the development of efficient vaccines is needed. Reverse genetics, allowing the introduction of targeted modifications into the viral genome and the production of attenuated live vaccine, may help to fight this rapidly spreading and emerging virus. It is routinely observed in farm trouts exposed to viral diseases that VHSV and IHNV coexist (26). By developing experimental coinfections by VHSV and IHNV in rainbow trout, Brudeseth et al. studied the pathogenesis and virus distribution (10). They found that both viruses established an infection and raised similar virus titers in kidneys, but the distribution of IHNV was more restricted in internal organs during the acute stage of the infection and was not detected in the brain. However, it generally is admitted that infection by one virus renders host cells resistant to a superinfecting virus.Superinfection exclusion, also known as homologous interference, is the phenomenon in which a cell infected with one type of virus or transfected with a viral replicon becomes resistant to a secondary infection with the same virus, whereas infection with unrelated viruses normally is unaffected (40, 51). Superinfection exclusion has been observed in a broad range of viruses, including vaccinia virus (14, 18), human immunodeficiency virus (HIV) (36, 37), vesicular stomatitis virus (VSV) (32, 43, 53), Borna disease virus (BDV) (24), measles virus (34), Sindbis virus (28), Semliki Forest virus (44), rubella virus (15), hepatitis C virus (HCV) (40, 51), and bovine viral diarrhea virus (BVDV) (31). Mechanisms of exclusion are diverse and have not been determined in all cases, but mechanisms described so far are caused by competition among different viruses for critical replicative pathways (for example, the use of the same receptors for the entry) or depend on the direct interaction of products of the primary infection with the secondary infecting virus. For example, the superinfection exclusion of VSV was found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: (i) a decreased rate of the formation of endocytic vesicles, (ii) a decreased rate of the internalization of receptor-bound ligands, and (iii) a competition with newly synthesized virus for the occupancy of coated pits (43). In contrast, the cytoplasmic accumulation of BDV nucleocapsid components appeared to prevent subsequent infection through a blockage of the polymerase activity of incoming viruses (24). Superinfection exclusion by BVDV was the result of dual mechanisms that were mediated by the structural protein E2, which blocks the entry of a homologous second virus, and by a blockage at the level of replication dependent on the level of primary viral RNA replication but not influenced by the expression of viral structural proteins, as observed for BDV (31). HIV employs its early gene product Nef to efficiently interfere with superinfection at the virus entry step by downregulating cell surface receptors (36). Finally, vaccinia virus expresses in newly infected cells two surface proteins that mark cells as infected and induce the repulsion of superinfecting viruses (18).In the present study, we described a reverse-genetics system for VHSV allowing the generation of a wild-type-like recombinant VHSV and a recombinant virus expressing a red fluorescent protein (Tomato). The system is based on the French strain 23/75 of VHSV, which is a hypervirulent and devastating strain for farmed rainbow trout belonging to genotype I (serotype III) and was isolated in France in 1975 from a brown trout (16, 23). Thus, this system provides a suitable starting point for identifying potential virulence determinants, as demonstrated by the deletion of the NV gene, and for developing attenuated derivatives as candidate vaccines. Using the available reverse-genetics system elaborated with IHNV, a recombinant IHNV expressing a green fluorescent protein (GFP) also was produced (8), and it was of interest to study whether a superinfection exclusion phenomenon could be observed between both VHSV and IHNV, whose cohabitation has been recorded often. We showed that up to 74% of a cell monolayer could be simultaneously infected by the viruses, demonstrating a limited viral interference between salmonid novirhabdoviruses and that, based on previous data, chimeric or pseudotyped viruses could be generated (6).     

Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.Maturation, by which a noninfectious immature virus particle is converted to an infectious virion, is an essential step in the replication cycle of many viruses. It often involves the proteolytic processing of a precursor protein coupled with the conformational transformation of the virion. The assembly pathway for flaviviruses is well established, and the structures of the entire virion as well as individual glycoproteins representing different stages of the life cycle have been determined. Thus, flaviviruses are an excellent system for investigating the dynamic aspects of maturation in enveloped viruses.Flavivirus maturation requires furin (21), a cellular protease located primarily in the trans-Golgi network (TGN) (16). Immature particles, containing heterodimers of the precursor membrane protein (prM) and the envelope protein (E), bud into the endoplasmic reticulum (ER) and then are transported through the cellular secretory pathway to the extracellular environment (12, 23). The cleavage of prM by furin generates the membrane-anchored protein M and the soluble product pr. In the mature, infectious virion, the pr peptide is absent, and the virus undergoes membrane fusion in the endosome at low pH. In contrast, immature particles produced from furin-deficient LoVo cells, or cells grown in the presence of protease inhibitors or acidotropic reagents to prevent furin cleavage, contain prM and are significantly less infectious (21).Crystal structures of the E protein (9, 14, 18, 19, 29) show that each polypeptide chain contains three domains: the structurally central amino-terminal domain (DI), the dimerization domain (DII) containing the fusion loop, and the carboxy-terminal immunoglobulin-like domain (DIII). In the presence of lipids, low pH induces rearrangements of the E proteins, resulting in the formation of homotrimers with the fusion loops and the C-terminal membrane anchors located at the same end (4, 15). The conformational change of the E proteins presumably facilitates the merging of the viral membrane with the endosomal membrane, thereby mediating the entry of virus through membrane fusion. The structures of the entire virion in the immature (26, 27) and the mature (10, 17, 25) forms have been studied by cryoelectron microscopy (cryoEM). The mature virion, approximately 500 Å in diameter, has a smooth surface on which 90 E dimers form a closely packed protein shell with a herringbone pattern (10). The immature particles (26, 27), with an external diameter of approximately 600 Å, contain 60 prominent spikes, each consisting of a trimer of prM-E heterodimers. The difference in the E protein organization is striking, indicating that the maturation process requires major positional exchanges of the E proteins (including their trans-membrane components). This raises the question of whether the structure of the immature form is physiologically relevant.Previously, we showed that at low pH, the immature virus undergoes a major conformational change in which the E proteins are rearranged into a configuration similar to that of the mature virus (24). The prM protein in the low-pH form of the immature virus, unlike the neutral-pH form, is accessible to furin cleavage (11, 24). Given that furin is abundant in the TGN, it is likely that the cleavage of the immature virus takes place in the TGN. However, it is not clear how virions are stabilized in the TGN after furin cleavage, where the pH value is similar to that of the endosome. It seems possible that pr remains associated with the virion in the TGN to prevent fusion, which is consistent with the observation that pr peptides comigrate with the virion in sucrose gradient sedimentation at pH 5.5 (24). However, in that experiment, approximately 30% of pr was found in fractions distinct from the virus (24). To further investigate the retention of pr at low pH, we report here the structure of furin-cleaved immature particles at pH 6.0 as determined by cryoEM. We show that at acidic pH the cleaved immature virus particles have essentially the same structure as that of the uncleaved immature particles, demonstrating that pr peptides are associated with virions at low pH and that furin cleavage by itself does not induce any major conformational changes. We further show that the presence of pr prevents the virus from interacting with liposomes at low pH, suggesting that pr retention inhibits membrane fusion in the TGN.