Electron Microscopic Characterization of the Defectiveness of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus Restricted in Assembly

The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39 degrees C) than either in cells grown at the permissive temperature (34 degrees C) or in wild-type MuLV-infected cells grown at 39 degrees C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34 degrees C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39 degrees C compared with those grown at 34 degrees C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.     

Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles.

The morphogenesis of hepatitis A virus (HAV) in BS-C-1 cells was examined by immunoblotting with antisera to capsid proteins and labeling of virus-specific proteins with L-[35S]methionine. Antiserum to VP2 detected two virus-specific proteins with apparent molecular masses of 30.6 and 30 kDa, representing VP0 and VP2, while antiserum to VP1 detected proteins with molecular masses of 33 and 40 kDa, representing VP1 and a virus-specific protein which we designated PX, respectively. Sedimentation of cell lysates revealed the presence of virions, procapsids, and pentamers, but particles analogous to the protomers of other picornaviruses were not detected. Although provirions and virions were not found as discrete species in our gradient system, it was evident that the rate of sedimentation was proportional to the relative amounts of VP0 and VP2 in particles, with slower-sedimenting particles (provirions) containing predominantly VP0 rather than VP2. Procapsids contained VP0 in addition to VP1 and VP3. Pentamers also contained VP0, but PX was present rather than VP1. These results suggest that PX is a precursor to VP1 and is most likely 1D2A. Primary cleavage of the viral polyprotein also occurs at the 2A-2B junction in cardioviruses and aphthoviruses, but assembly of pentamers containing 1D2A has not been reported for those viruses. The absence of detectable levels of protomers suggests a high efficiency of pentamer formation, which may be related to the high efficiency of viral RNA encapsidation for HAV (D.A. Anderson, B.C. Ross, and S.A. Locarnini, J. Virol. 62:4201-4206, 1988). The results of this study reveal further unusual aspects of the HAV replicative cycle which distinguish it from other picornaviruses and may contribute to its restricted replication in cell culture.     

Studies of two temperature-sensitive mutants of Mengo virus.

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA- ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 degrees C) to the nonpermissive (39 degrees C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA- phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 degrees C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant. Subviral (53S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 degrees C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.     

Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity.

Experiments done with a temperature"sensitive mutant of herpes simplex virus type 1 (HSV-1) have revealed that one of the virisn glycoproteins, designated VP7(B2), is apparently not required for the production of enveloped virus particles, whereas it does play a critical role in virion infectivity. The mutant, designated HSV-1[HFEM]tsB5, fails to accumulate VP7(B2) at nonpermissive temperature and produces virions that lack detectable quantities of this glycoprotein and that have very low specific infectivity. The poor infectivity of the virions is most readily explained by failure of penetration into the host cell rather than by failure of adsorption to cells because it was shown that the VP7(B2)-deficient virions can bind to cells and that polyethylene glycol, an agent known to promote membrane fusion, can significantly enhance infectivity of the adsorbed virions.     

A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KDeltaUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KDeltaUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KDeltaUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KDeltaUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KDeltaUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.     

A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.     

Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C(pro). The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C(pro) to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C(pro)-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C(pro) and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C(pro)-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.     

Synthesis and assembly of hepatitis A virus-specific proteins in BS-C-1 cells.

To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35S]methionine and L-[35S]cysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VP0, VP2, and VP3; procapsids contained VP1, VP0, and VP3; and pentamers contained PX, VP0, and VP3, as previously shown by immunoblotting (D.A. Anderson and B.C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1- and VP2-reactive sequences and sedimenting slightly more slowly than 14S pentamers, which may represent intact P12A assembled into pentamers as has been reported for the P1 of some other picornaviruses (S. McGregor and R. R. Rueckert, J. Virol. 21:548-553, 1977). The results of this study suggest that cleavage of the hepatitis A virus polyprotein to produce pentamers is protracted (though not rate limiting) early in infection, while the assembly of pentamers into higher structures is a rapid process once sufficient viral RNA is produced for encapsidation.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.     

Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants.

The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII.     

Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity.

Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.     

Characterization of a Temperature-Sensitive Fiber Mutant of Type 5 Adenovirus and Effect of the Mutation on Virion Assembly

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5°C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32°C, fiber polypeptides synthesized at 39.5°C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5°C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32°C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.