A poliovirus-susceptible transgenic mouse model as a possible replacement for the monkey neurovirulence test of oral poliovirus vaccine

Two poliovirus-susceptible transgenic mouse (Tg PVR) strains, Tg1 and Tg21, were compared with the monkey test for their sensitivity to neurovirulence of live oral poliovirus vaccine (OPV). Intracerebral (i.c.) and intraspinal (i.s.) routes of inoculation were investigated to determine the most suitable combination of mouse strain and route. Evaluation of the mouse tests was performed using several indicators; clinical score and failure time were selected as the most efficient. Tg1 and Tg21 mice inoculated i.s. with type 2, and Tg21 mice inoculated i.s. with type 3 OPV were determined to be the most appropriate systems, whereas they are shown not to be suitable for type 1 OPV. The sensitivity of each of the two mouse models was at least equal to that of the monkey test, suggesting that these mouse systems might be considered as a potential replacement for the monkey test of OPV. However, more data are needed to establish regulatory criteria of acceptability for vaccine lots tested in Tg PVR mice. This is the first study conducted with Tg PVR mice with all three types of poliovirus vaccine preparations.     

Interaction of poliovirus with its receptor affords a high level of infectivity to the virion in poliovirus infections mediated by the Fc receptor

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcgamma receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.     

Regulation of lymphocyte migration into lymph nodes by high endothelial venules

The transgenic (Tg) mice carrying the human gene for poliovirus receptor (PVR) are susceptible to poliovirus intravenously (i.v.) inoculated as well as intracerebrally or intraspinally inoculated. Thus, i.v.-inoculated poliovirus may invade the central nervous system (CNS) through the blood-brain barrier (BBB). To know the contribution of PVR to tissue distribution and BBB permeability of i.v.-inoculated polioviruses, these dissemination processes were investigated and compared between the Tg mice and non-Tg mice. Distribution profile of i.v.-inoculated poliovirus in various tissues of the Tg mice is similar to that in non-Tg mice. The data suggest that tissue distribution of the virus occurs independently of the transgene for PVR. The amount of poliovirus delivered to the CNS suggested the existence of a specific delivery system of the virus to the CNS. Virus accumulation in the CNS of the Tg mice was measured up to 7.5 hr after the i.v. inoculation. The viruses, regardless of whether the virulent or attenuated strain, seem to accumulate at a constant rate of approximately 0.2 microliter/min/g tissue. Similar phenomena were observed when the viruses were inoculated into non-Tg mice. The rates of the virus accumulation in the CNS are more than 100 times higher than that of albumin, which is considered not to permeate through the BBB via a specific transport system, and only three times lower than that of monoclonal antibody against transferrin receptor (OX-26), which is a potential candidate as a drug delivery vehicle specific to the CNS. These data suggest that polioviruses permeate through the BBB at a fairly high rate, independently of PVR and virus strains.     

One of two NTP binding sites in poliovirus RNA polymerase required for RNA replication

The poliovirus RNA-dependent RNA polymerase (3Dpol) has been shown to contain two NTP binding sites by chemical cross-linking of oxidized nucleotide to the intact protein. Only one site (Lys-61) was shown to be essential for RNA chain elongation activity by purified enzyme; however, a full-length viral RNA, coding for an altered lysine residue (K276L) in the second site, generated virus with a minute plaque phenotype that rapidly reverted to a wild-type phenotype with Arg-276 replacing Leu-276 in 3D. Viruses with lysine to leucine substitutions in other positions of the second binding site of their polymerase proteins grew with wild-type phenotype. To test the significance of the second binding site, poliovirus 3Dpol was generated with lysine (wild-type), leucine, or arginine at residue 276 and tested for NTP cross-linking using 32P-oxidized GTP. Analysis of cyanogen bromide peptides of each 3D preparation showed that the second NTP binding site had severely reduced NTP binding in mu276(Leu) but not in the revertant mu276(Arg), despite the reported requirement for lysine in the cross-linking reaction. To eliminate the possibility that 32P-oxidized GTP cross-linked to Arg at residue 276, a model system was designed with unmodified amino acid or acetylated (alpha-amino) amino acid and 32P-oxidized GTP. Cross-linking to lysine, but not leucine or arginine, was observed thus eliminating the possibility that NTP could be cross-linked to residue 276 in 3D. We conclude that NTP binding at the second site in poliovirus 3D is at lysine residues at positions other than 276 (278 or 283), and nucleotide binding at these sites has no bearing on elongation activity or replication of the virus. Nucleotide binding only at the site including Lys-61 is essential for RNA replication.     

Comparison of replication rates and pathogenicities between the SA14 parent and SA14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons

The replication rates and pathogenicities of the SA 14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis (JE) virus in neurons of the mouse brain following intracerebral inoculation were compared. All the mice inoculated with the SA 14 parent strain died within one week postinoculation (p.i.), whereas all the mice inoculated with the SA 14-14-2 vaccine strains survived without showing any signs of central nervous system (CNS) involvement. The virus titers of the mouse brains inoculated with the SA 14 strain reached progressively higher levels until day 5 when the animals died. On the other hand, the virus titers of the mouse brains inoculated with the SA 14-14-2 strain persisted at low levels for several days and could not be detected after 10 days. In the routine electron microscopical study, a majority of neurons in the mouse brains inoculated with the SA 14 strain contained virions and showed characteristic cytopathological changes in connection with viral replication. In the brains inoculated with the SA 14-14-2 strain, however, we failed to find neurons containing virions or showing characteristic cytopathological changes. In the alkaline phosphatase immunostaining of paraffin-embedded sections, a majority of neurons in the brains of mice inoculated with the SA 14 strain stained positively on day 5 p.i., but only a small number of neurons in scattered small foci stained positively in the brains inoculated with the SA 14-14-2 strain. The immunogold staining of Vibratome sections also revealed the identical patterns; moreover, electron microscopical examination of the immunopositive foci of the brain inoculated with the vaccine strain revealed neurons that contained virions in dilated cisternae of rough endoplasmic reticulum (RER), indicating that the SA 14-14-2 strain also replicated, albeit poorly, in neurons. The present results showed that upon intracerebral inoculation into mice the SA 14 parent strain of JE virus grew vigorously in a large number of neurons, killing the animals, while the SA 14-14-2 vaccine strain grew poorly only in a small number of neurons without causing mortality. Possible mechanisms involved in the alteration of pathogenicity between the SA 14 parent virus and the SA 14-14-2 vaccine virus are discussed.     

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.     

Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of three poliovirus serotypes

BACKGROUND: Polioviruses are human pathogens and the causative agents of poliomyelitis. Polioviruses are icosahedral single-stranded RNA viruses, which belong to the picornavirus family, and occur as three distinct serotypes. All three serotypes of poliovirus can infect primates, but only type 2 can infect mice. The crystal structures of a type 1 and a type 3 poliovirus are already known. Structural studies of poliovirus type 2 Lansing (PV2L) were initiated to try to enhance our understanding of the differences in host range specificity, antigenicity and receptor binding among the three serotypes of poliovirus. RESULTS: The crystal structure of the mouse neurovirulent PV2L complexed with a potent antiviral agent, SCH48973, was determined at 2.9 A resolution. Structural differences among the three poliovirus serotypes occur primarily in the loop regions of the viral coat proteins (VPs), most notably in the loops of VP1 that cluster near the fivefold axes of the capsid, where the BC loop of PV2L is disordered. Unlike other known structures of enteroviruses, the entire polypeptide chain of PV2L VP4 is visible in the electron density and RNA bases are observed stacking with conserved aromatic residues (Tyr4020 and Phe4046) of VP4. The broad-spectrum antiviral agent SCH48973 is observed binding in a pocket within the beta-barrel of VP1, in approximately the same location that natural 'pocket factors' bind to polioviruses. SCH48973 forms predominantly hydrophobic interactions with the pocket residues. CONCLUSIONS: Some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice may occur in the vicinity of the fivefold axis of the poliovirus, where there are significant structural differences among the three poliovirus serotypes in the surface exposed loops of VP1 (BC, DE, and HI). A surface depression is located at the fivefold axis of PV2L that is not present in the other two poliovirus serotypes. The observed interaction of RNA with VP4 supports the observation that loss of VP4 ultimately leads to the loss of viral RNA. A model is proposed that suggests dual involvement of the virion fivefold and pseudo-threefold axes in receptor-mediated initiation of infection by picornaviruses.     

The origin of congenital cholesteatoma

Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GMI-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GMI-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.     

Mouse adenovirus type-1 replication is restricted to vascular endothelium in the CNS of susceptible strains of mice

Previous studies have shown that mouse adenovirus type-1 (MAV-1) caused a fatal hemorrhagic encephalitis in certain strains of mice. C57BI/6 mice exhibited 100% mortality when given as little 10(3) plaque-forming units (PFU) of MAV, in contrast to BALB/c mice which were resistant to as many as 10(6) PFU. Susceptible animals died with a flaccid paralysis on the 3rd or 4th day after inoculation. The brains and spinal cords of these animals displayed numerous petechial hemorrhages that were found in virtually all areas of the brain, but were more numerous in white matter. In this paper, immunohistochemistry and electron microscopy were used to identify the viral target of replication within the CNS of susceptible mice. These studies showed that the CNS vascular endothelial cell was the primary site of viral replication within the CNS of mice infected with MAV-1. Characterization of cytokine mRNA levels and disease course in immunodeficient mice revealed that the host immune response played little, if any, role in the pathogenesis of MAV-1 disease in susceptible mice and was not responsible for the resistance of BALB/c mice. These results support the conclusion that disease course and outcome in susceptible and resistant strains of mice were determined primarily by the ability of the virus to replicate within the CNS vascular endothelium.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

The poliovirus empty capsid specifically recognizes the poliovirus receptor and undergoes some, but not all, of the transitions associated with cell entry

Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled.     

Regulated expression of the interferon-induced protein kinase p68 (PKR) by vaccinia virus recombinants inhibits the replication of vesicular stomatitis virus but not that of poliovirus

A direct antiviral role of the interferon-induced human protein kinase p68 has been shown only against encephalomyocarditis virus (EMCV) and vaccinia virus (VV). To determine if p68 kinase (PKR) has a broad antiviral effect, we have used coinfections between VV recombinants expressing p68 kinase under regulation of the lac I operator/repressor elements of Escherichia coli and two RNA viruses, vesicular stomatitis virus (VSV) and poliovirus. In cells coinfected with VV recombinants and VSV, induction with isopropyl-B-D-thiogalactoside (IPTG) of wild-type p68 kinase or a mutant lacking the dsRNA binding domain resulted in inhibition of both VV and VSV protein synthesis. This inhibition is not observed in cells infected with a catalytically inactive point mutant lys-arg296 of p68 kinase. When cells are coinfected with VV recombinants and poliovirus, induction of active p68 kinase resulted in a decrease in VV proteins but not in poliovirus proteins or poliovirus yields. Immunoblot analysis revealed that p68 kinase was expressed during mixed infections. Our results demonstrate a differential effect of p68 kinase on the replication of VV, VSV, and poliovirus. We suggest that in a particular virus-cell system, the different sensitivity of a virus to p68 kinase is probably due to levels of active enzyme.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii

The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HAI in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAI. BAG-5 positive organisms were not seen 2-5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HAI but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HAI and more consistently at 48 HAI. Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.     

Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase

Poly(rC) binding protein 2 (PCBP2) forms a specific ribonucleoprotein (RNP) complex with the 5'-terminal sequences of poliovirus genomic RNA, as determined by electrophoretic mobility shift assay. Mutational analysis showed that binding requires the wild-type nucleotide sequence at positions 20-25. This sequence is predicted to localize to a specific stem-loop within a cloverleaf-like secondary structure element at the 5'-terminus of the viral RNA. Addition of purified poliovirus 3CD to the PCBP2/RNA binding reaction results in the formation of a ternary complex, whose electrophoretic mobility is further retarded. These properties are consistent with those described for the unidentified cellular protein in the RNP complex described by Andino et al. (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598). Dicistronic RNAs containing mutations in the 5' cloverleaf-like structure of poliovirus that abate PCBP2 binding show a decrease in RNA replication and translation of gene products directed by the poliovirus 5' noncoding region in vitro, suggesting that the interaction of PCBP2 with these sequences performs a dual role in the virus life cycle by facilitating both viral protein synthesis and initiation of viral RNA synthesis.     

Oral inoculation of SCID mice with an attenuated herpes simplex virus-1 strain causes persistent enteric nervous system infection and gastric ulcers without direct mucosal infection

BACKGROUND: After placement of herpes simplex virus type 1 (HSV-1) into the esophageal lumen of BALB/c mice, the virus replicated in enteric neurons within the esophagus and stomach and was transported to the sensory ganglia of the vagus nerve (nodose ganglia), where viral replication also occurs and where ultimately a long term latent infection is established. This described infection of immunocompetent mice primarily involved neuronal cells and associated satellite cells. EXPERIMENTAL DESIGN: Severe combined immunodeficient (SCID) mice were orally infected with an attenuated strain of HSV-1 to better identify sites of viral involvement in the gastrointestinal tract, particularly the mucosa. RESULTS: Three to five weeks after oral inoculation of SCID mice with HSV-1 strain in1814, a persistent viral infection of the gastrointestinal tract was established in most of the mice. Extensive viral replication was detected by immunohistochemistry throughout pathways of the vagus nerve and within the intrinsic enteric nervous system. Despite this ultimately fatal infection, viral replication in the gut occurred almost exclusively in enteric neurons and their processes; viral proteins were occasionally seen in smooth muscle cells immediately adjacent to heavily infected enteric ganglia. More than 50% of these persistently infected mice, when killed 18 to 31 days postinoculation, had gastric ulcers that were identified grossly and histologically. Only one of the 40 gastric ulcers was found to contain viral Ag. The remaining ulcers, although devoid of viral proteins, were found adjacent to virus-infected ganglia. CONCLUSIONS: HSV-1 can enter enteric neurons with minimal initial mucosal involvement, and once inside the nervous system, the virus is contained there despite the absence of a specific host immune response. Furthermore, chronically infected enteric neurons may provide an indirect mechanism for the pathogenesis of gastric ulcers in these immune-deficient mice.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.