Virus Replication in Enucleate Cells: Vesicular Stomatitis Virus and Influenza Virus

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.     

Inhibition of Protein Synthesis in L Cells Infected with Vesicular Stomatitis Virus

The inhibition of protein synthesis in L cells by vesicular stomatitis virus (VSV) requires the synthesis of new protein subsequent to virus infection. However, two mechanisms may be involved in the inhibition of cell protein synthesis by VSV: an initial, multiplicity-dependent, ultraviolet-insensitive inhibition and a progressive, ultraviolet-sensitive inhibition.     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.     

Vaccination with a Recombinant Vesicular Stomatitis Virus Expressing an Influenza Virus Hemagglutinin Provides Complete Protection from Influenza Virus Challenge

Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982–5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.     

Protein Kinase and Phosphoproteins of Vesicular Stomatitis Virus

Protein kinases of similar but not identical activity were found associated with vesicular stomatitis (VS) virions grown in mouse L cells, primary chicken embryo (CE) cells, and BHK-21 cells, as well as being present in VS virions grown in HeLa and Aedes albopictus cells. The virion kinase preferentially phosphorylated the nucleocapsid NS protein in vitro and to a lesser extent the envelope M protein. Other virion proteins were phosphorylated in vitro only after drastic detergent treatment. Partial evidence that the virion kinase is of cellular origin was obtained by finding reduced enzyme activity in virions released from cells pretreated with actinomycin D and cycloheximide. Selective detergent and detergent-salt fractionation of VS virions revealed that the kinase activity was present in the envelope but not the spikes. The virion kinase activity in a Triton-salt-solubilized envelope fraction could be separated from M and G proteins and partially purified by phosphocellulose column chromatography. Virions released from L, CE, and BHK-21 cells infected in the presence of [(32)P]orthophosphate were labeled almost exclusively in the NS protein. Both soluble and nucleocapsid-associated NS phosphoprotein were present in cytoplasmic extracts of VS viral-infected L cells. The origin and function of the NS phosphoprotein remain to be elucidated.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

Fusion of Vesicular Stomatitis Virus with the Cytoplasmic Membrane of L Cells

At an early stage in infection, vesicular stomatitis viruses were attached to the surface of L cells by fusion of the viral and cell membranes.     

Proteomic Analyses of Gastric Cancer Cells Treated with Vesicular Stomatitis Virus Matrix Protein

Gastric cancer constitutes the second leading cause of mortality worldwide and the fourth most common cancer. While chemotherapy remains the primary treatment for both resectable and advanced gastric cancer, most gastric cancers are naturally resistant to anticancer drugs, rendering new therapeutic avenues in dire need. Vesicular stomatitis virus (VSV) was proved to preferentially replicate in many types of tumor cells and eventually induce apoptosis of host cells. The vesicular stomatitis virus matrix protein (MP) plays a major role in its effects. This study proved that expression of MP could effectively inhibit proliferation and induce cell death in gastric carcinoma MKN28 cells. Furthermore, we utilized a proteomics strategy to characterize proteome-wide alterations between MP-treated MKN28 lines and their untreated counterparts. A total of 97 spots were positively identified as differentially expressed, and of these 62 proteins were up-regulated, whereas 35 proteins were down-regulated. Functional analysis unraveled three significantly modified gene product subgroups: glycolytic enzymes, reactive oxygen species-associated proteins and the proteins regulating RNA transport and maturation. Expression of three altered proteins was further validated by semi-quantitative RT-PCR or/and western blotting. Furthermore, we demonstrated that MP expression could induce rapid intracellular ROS accumulation in MKN28 cells. These results provide evidence for the anti-cancer potential of MP, and a novel MP-mediated apoptotic signaling pathway is proposed. Our findings are considered a significant step toward a better understanding the mechanism of MP-induced anti-cancer effect.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Comparison of Membrane Protein Glycopeptides of Sindbis Virus and Vesicular Stomatitis Virus

A comparison has been made of the membrane glycoproteins and glycopeptides from two enveloped viruses, Sindbis virus and vesicular stomatitis virus (VSV). Glycopeptides isolated from Sindbis virus and VSV grown in the same host appear to differ principally in the number of sialic acid residues per glycopeptide; when sialic acid is removed by mild acid treatment, the glycopeptides of the two viral proteins are indistinguishable by exclusion chromatography. Preliminary evidence argues that the carbohydrate moiety covalently bound to different virus-specified membrane proteins may be specified principally by the host.     

Combined Administration with DNA Encoding Vesicular Stomatitis Virus G Protein Enhances DNA Vaccine Potency

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8⁺ T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)—together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen—elicited robust E7-specific CD8⁺ T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.Due to their safety, low manufacturing cost, and ease of production, DNA vaccines have emerged as one of the most attractive approaches to prevent and treat viral infections, as well as cancers with defined tumor-associated antigens. Several studies have demonstrated that DNA vaccines can produce significant CD8⁺ T-cell-mediated immunity—which is often essential in the elimination of pathogens and malignancies—together with therapeutic benefit in various animal models of disease (4, 21-23, 29). However, DNA vaccines have achieved limited success in the clinic, since they generally elicit limited CD8⁺ T-cell responses in humans despite repeated high-dose administration (15, 28). Therefore, there is a critical need to develop DNA vaccines which generate these types of responses.DNA vaccines are commonly delivered by inoculation into skeletal muscle, where myocytes uptake them and express their encoded antigen. Myocytes, however, lack major histocompatibility complex (MHC) class II and costimulatory molecules and, as a result, have poor ability to prime naive T cells. Thus, the potency of DNA vaccines depends on sequential phases of antigen transfer from myocytes to sentinel dendritic cells (DCs), followed in close succession by the maturation of these DCs. The DCs then migrate to lymphoid organs, where they can present the antigen to and activate cognate naive CD8⁺ T cells (11). In the absence of either of these phases, either a null or a tolerogenic immune response is generated.One of the major challenges to the generation of CD8⁺ T-cell responses is therefore the design of vaccines that efficiently target antigen to and stimulate the maturation of DCs in a concerted manner. We hypothesized that this could be accomplished through the induction of an inflammatory form of myocyte death with concentrated antigen release to DCs upon DNA immunization. Viral fusogenic membrane glycoproteins (FMGs) represent ideal agents for eliciting such an effect. It has been shown that FMGs induce the fusion of cells into large multinucleated syncytia, which are lysed rapidly by nonapoptotic mechanisms (1) and in the process shed exosomes from the membrane (2). We reasoned that, since nonapoptotic death has been linked to stimulation of the innate immune system (25) and since exosomes have been reported to contain antigen that can be readily taken up by DCs (30), FMGs may bolster the release of antigens encoded by the DNA vaccine, leading to enhanced antigen-specific CD8⁺ T-cell responses.We have previously created and characterized a panel of DNA vaccines targeting the E7 oncoprotein of human papillomavirus (HPV) type 16, a model cervical cancer antigen. Because E7 is required for the maintenance of the transformed cellular phenotype and is expressed in virtually all cases of cervical cancers, it provides an ideal molecular target against which to apply our vaccination approach. In head-to-head comparisons of these vaccines, we showed that one particular construct, CRT/E7, could generate the most potent immunological and therapeutic effect against E7-expressing tumors (14). However, although CRT/E7 can cure mice with small tumors, it invariably fails to control more advanced forms of disease. In light of this obstacle, we sought to determine whether VSV-G could render CRT/E7 therapeutically effective against otherwise refractory E7-expressing tumors. We have previously characterized the growth kinetics of one such aggressive tumor model, TC-1, in mice and observed consistently that 1 week after subcutaneous challenge with 5 × 10⁵ cancerous cells, the mass becomes clearly detectable by palpation and visual inspection, and vaccination with CRT/E7—even with repeated high dose administration—has negligible beneficial effect on disease progression or animal survival. At this point, we considered the cancer to be sufficiently advanced, and this timeline was therefore adopted for the tumor treatment experiments in the present study.In the present study, we explored whether combined administration with VSV-G could successfully control the growth of aggressive TC-1 tumors refractory to treatment with CRT/E7 alone, and potential mechanisms by which this might be achieved. Collectively, our data indicate that incorporation of FMGs into DNA vaccines represents a therapeutically promising strategy for the generation of antigen-specific T-cell-mediated immunity against viral infections and cancer.     

Incorporation of L Cell Sterols into Vesicular Stomatitis Virus

The incorporation of host cell sterol into vesicular stomatitis virus can be effectively studied in an L cell system. The end product of de novo sterol synthesis in the L cell is desmosterol, and as the concentration of cholesterol in the medium is increased the cells incorporate the exogenous cholesterol and the synthesis of desmosterol decreases. L cells which contained desmosterol as their sole sterol produced virus whose sterol content was similarly composed of only desmosterol. Virus grown in L cells which had a constantly changing sterol ratio also contained a mixture of cholesterol and desmosterol, but the virus was found to be more enriched in cholesterol than in the L cells in which it was grown. Viral stability, growth, and plaquing efficiency were tested and found not to be affected by the alteration of its sterol composition, i.e., by partially or completely replacing cholesterol with desmosterol.     

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

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Protein Composition of the Structural Components of Vesicular Stomatitis Virus

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Alterations in the Structure of the Oligosaccharide of Vesicular Stomatitis Virus G Protein by Swainsonine

Swainsonine, an inhibitor of glycoprotein processing, inhibits the formation of the normal oligosaccharide chain of the G protein of vesicular stomatitis virus. Thus, when vesicular stomatitis virus was grown in baby hamster kidney cells in the presence of swainsonine (15 to 500 ng/ml) and labeled with [2-(3)H]mannose, the oligosaccharide portion of the G protein was completely susceptible to the action of endoglucosaminidase H. However, the normal viral glycoprotein is not susceptible to this enzyme. Various enzymatic treatments and methylation studies of the mannose-labeled oligosaccharides suggest that swainsonine causes the formation of a hybrid-type oligosaccharide having an oligomannosyl core (Man(5)GlcNAc(2)-Asn) characteristic of neutral oligosaccharides plus the branch structure (NeuNAc-Gal-GlcNAc) characteristic of the complex oligosaccharides. A structure for this hybrid oligosaccharide is proposed. Swainsonine had no effect on the incorporation of [(14)C]leucine into viral proteins, nor did it change the number of PFU produced in these cultures. It did, however, slightly decrease the incorporation of [(3)H]glucosamine and increase the incorporation of [(3)H]mannose. Vesicular stomatitis virus raised in the presence of swainsonine bound much more tightly to columns of concanavalin A-Sepharose than did control virus. Swainsonine had to be added within the first 4 or 5 h of virus infection to be effective. Thus, when 100 ng of the alkaloid per ml was added at any time within the first 3 h of infection, essentially all of the glycoprotein was susceptible to digestion by endoglucosaminidase H. However, when swainsonine was added 4 h after the start of infection, 30% of the glycopeptides became resistant to endoglucosaminidase H; at 5 h, 70% were resistant. The effect of swainsonine was reversible since removal of the alkaloid allowed the cells to form the normal complex glycoproteins. However, the time of removal was critical in terms of oligosaccharide structure.