Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.     

Stereo images of vesicular stomatitis virus assembly.

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.     

The nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane.

Bacteriophage phi 6 infects its host, the Gram-negative bacterium Pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane. In this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle. This indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously expected. Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells. The penetration is dependent on the energized state of the host cytoplasmic membrane. About 25% of the entering core particles are re-used in the progeny viruses.     

Intracellular hepadnavirus nucleocapsids are selected for secretion by envelope protein-independent membrane binding

Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surface-exposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBV-transfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.     

Expression, purification and characterization of full-length RNA-free hepatitis B core particles

The nucleocapsid or core particle of the hepatitis B virus has become one of the favourite recombinant vaccine carriers for foreign peptides, proteins and stimulatory oligonucleotides. The core protein consists of three regions: an N-terminal, a central and a C-terminal region that can accommodate the addition or insertion of the foreign sequences. The protamine-like C-terminal region that binds host RNA randomly during recombinant particle formation is often truncated. It is commonly thought that these truncations do not affect particle assembly. Recent studies have demonstrated that the C-terminal domains mediate a glycosaminoglycan-dependent attachment of nucleocapsids to the plasma membranes of host cells. This interaction might well contribute to the immunogenicity of nucleocapsids. Testing the hypothesis that full-length particles might be safer and superior for the induction of an immune response against the nucleocapsids and inserted sequences, requires the availability of purified particles. In this report, we detail a novel method for the synthesis and purification of full-length core particles essentially free of RNA from Escherichia coli.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

M-X-I motif of semliki forest virus capsid protein affects nucleocapsid assembly

Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids.     

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

Ultrastructural observations in hepatitis C virus-infected lymphoid cells

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.     

Hepatitis C virus core protein is efficiently released into the culture medium in insect cells

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.     

Effect of cAMP-dependent protein kinase A (PKA) on HCV nucleocapsid assembly and degradation.

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein,production of the viral particle and expression of host proteins involved in pathogenicity

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C‐terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin‐like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti‐HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti‐HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP‐dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV‐infected cells was significantly impaired by LY411575 in a dose‐dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir‐dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV‐related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.     

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

A genetic interaction between the core and NS3 proteins of hepatitis C virus is essential for production of infectious virus

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1(T)-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1(T)-64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1(T)-64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.     

Viral Persistence, Antibody to E1 and E2, and Hypervariable Region 1 Sequence Stability in Hepatitis C Virus-Inoculated Chimpanzees

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.     

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.