Envelope Proteins of Vesicular Stomatitis Virus: Effect of Temperature-Sensitive Mutations in Complementation Groups III and V

All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations.     

Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly.

The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.     

Cytoplasmic Domain of Sendai Virus HN Protein Contains a Specific Sequence Required for Its Incorporation into Virions

In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.     

Kinetics of Incorporation of Structural Proteins into Sindbis Virions

The morphogenesis of Sindbis virus was studied by determining the kinetics with which newly synthesized nucleocapsid and envelope proteins appeared in virions released into the extracellular medium. Assembly of the nucleocapsid was more rapid than modification of the cellular membrane by the addition of the viral envelope protein. However, both viral structural proteins were efficiently incorporated into virions; a 0.5-hr pulse-labeling period resulted in the release of maximally labeled virus during the next hour. When protein synthesis was inhibited, release of virus soon declined even though large amounts of both viral structural proteins were present within the cell and ribonucleic acid replication was unaffected.     

Time Course of Synthesis and Assembly of Influenza Virus Proteins

The synthesis of viral polypeptides was analyzed in BHK-21-F cells infected with the WSN strain of influenza virus at various times in the growth cycle. The relative amounts of polypeptides P, HA, NP, and NS did not change markedly between early and late times in the growth cycle; however, there was a progressive increase in the relative amount of the M polypeptide at later time points. In cell fractionation experiments, the patterns of newly synthesized polypeptides associated with various cytoplasmic fractions remained similar throughout the growth cycle except for an increase in polypeptide M in all fractions late in the growth cycle. The HA polypeptide was chased out of cytoplasmic membranes completely 6 h after synthesis, whereas the M polypeptide was not chased effectively from such membranes. Marked differences were found in the incorporation into mature virions of polypeptides synthesized at different times in the growth cycle. Polypeptides P and NP synthesized at early times were incorporated preferentially, whereas M was synthesized and incorporated predominantly late in the growth cycle. The fact that the rates of incorporation of polypeptides into virions differed significantly from their rates of synthesis indicates that different polypeptides were assembled into virions by distinct pathways.     

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Production of Uninfectious Human Immunodeficiency Virus Type 1 Containing Viral Protein R Fused to a Single-Chain Antibody against Viral Integrase

A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity.     

Mathematical analysis of the oncornavirus maturation process (virion RNA conversion and morphological condensation)

The rate of the maturation process of avian myeloblastosis virus experimentally estimated on the basis of genomic viral RNA conversion and morphological transition of virions was mathematically analysed. Three mathematical models were suggested and fitted to experimental data. It was found that: (a) model of simple kinetics (Model 1) does not agree with experimental data. Therefore, two hypotheses were considered in further mathematical modelling: (b) virions are identical in time of budding: maturation is dependent on the presence of a virion component which is degraded with time (Model 2). This model agrees with experimental data in all stages of the maturation process. (c) Virions are released from cells at different stages of assembly (Model 3). This model differs from experimental data especially in early stages of maturation. The hypothesis used for the construction of Model 2 seems to be the most plausible to explain the maturation process and is in agreement with data of murine leukemia virus maturation which was found to be accomplished by cleavage of p70 precursor protein.     

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.     

Temporal morphogenesis of herpes simplex virus type 1-infected and brefeldin A-treated human fibroblasts

BACKGROUND: Insights in the herpesvirus-cell interactions are of general cell biology interest, especially to studies of intracellular transport, and of considerable significance in the efforts to generate drugs, vaccines, and gene therapy. However, the pathway of virus particle egress and maturation is a contentious issue. MATERIALS AND METHODS: The intracellular transport was inhibited in cultured herpes simplex virus type 1 (HSV-1) infected human fibroblasts by brefeldin A (BFA). The virus-cell interactions including the viral envelopment, transport of HSV-1 virions, and transport of viral glycoprotein D (gD-1) and glycoprotein C (gC-1) were studied by titration assay, immunoblot, immunofluorescence light microscopy, and immunogold electron microscopy of cryosections. RESULTS: gD-1 and gC-1 were synthesized and normally transported to the plasma membranes of untreated HSV-1 infected host cells. BFA (1 microg/ml medium) effectively blocked the transport of the glycoproteins to the plasma membranes and affected the tubulin and vimentin of the cytoskeleton. Viral particles and glycoproteins accumulated in the perinuclear space and the endoplasmic reticulum of BFA treated cells. Withdrawal of BFA influence up to 9 hr resulted in restored tubulin and vimentin, transport of glycoproteins to the plasma membranes, and steady release of infectious viral particles to the extracellular space superior to the cellular assembly of new virions. The ultrastructural data presented support that the primary envelopment of viral particles occur at the nuclear membranes containing immature glycoproteins followed by multiple de-envelopments and re-envelopments of the virions during the transport and maturation in the endoplasmic reticulum and the Golgi complex. CONCLUSIONS: BFA-induced changes include the cytoskeleton with significant effect on HSV-1 maturation and egress. The data support a multiple-step envelopment of HSV-1 in a common pathway of glycoprotein synthesis and virion egress.     

LPP-1 Infection of the Blue-Green Alga Plectonema boryanum: I. Electron Microscopy

One-step growth and intracellular growth experiments were performed at high multiplicities of the virus LPP-1 during the infection of the blue-green alga Plectonema boryanum. The eclipse period lasts until 4 hr after infection, the latent period terminates at 6 hr, and the rise period continues until 14 to 16 hr after infection. The burst size was independent of multiplicity of infection over the ranges from 1 to 50. The burst size was 3,000 to 5,000 plaque-forming units (PFU) per infectious center or about 200 PFU per cell. Samples for electron microscopy were taken at characteristic times during the lytic cycle. The first sign of viral infection was the invagination of the photosynthetic lamellae at 3 hr after infection. Mature virions were visible at 4 hr. By 6 to 7 hr, many mature intracellular viral particles could be seen, with lysis beginning at 7 hr. By 10 hr after infection, all infected cells contained mature virions. No evidence for mass migration of performed viral precursors was obtained. The invagination of the lamellae could be prevented by the early addition of chloramphenicol, which implies that this process requires protein synthesis.     

Translation of avian myeloblastosis virus RNA in a reticulocyte cell-free system.

AMV-RNA was translated into a precursor polypeptide of 76,000–80,000 daltons in a reticulocyte cell-free system. Besides this high molecular weight precursor, several smaller precursor polypeptides and the four major internal structural viral proteins were also synthesized. These virus-specific translation products were detectable after immunoprecipitation with antiserum against the gs-antigens of AMV.     

Growth and Maturation of a Vesicular Stomatitis Virus Temperature-Sensitive Mutant and Its Central Nervous System Isolate

A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31, produces a prolonged central nervous system disease in mice with pathological features similar to those of slow viral diseases. tsG31 and the subsequent virus recovered from the central nervous system (tsG31BP) of mice infected with tsG31 were compared with the parental wild-type (WT) VSV for plaque morphology, growth kinetics, thermal sensitivity of the virions, and viral protein synthesis and maturation. Several properties of the central nervous system isolate distinguished this virus from the original tsG31 and the WT VSV. The WT VSV produced clear plaques with complete cell lysis, and the tsG31 produced diffuse plaques and incomplete cell lysis, whereas the tsG31BP had clear plaques similar to those of the WT VSV. Although plaque morphology suggested that tsG31BP virus was a revertant to the WT, growth kinetics in either BHK-21 or neuroblastoma (N-18) cells indicated that this virus was similar to tsG31, with a productive cycle at 31 degrees C and no infectious virus at 39 degrees C. At 37 degrees C, however, the tsG31BP matured much slower than did the original tsG31 (and produced only 1% of the yield measured at 31 degrees C). WT VSV produced similar quantities of infectious virions at 31, 37, and 39 degrees C. The lack of infectious virions at 39 degrees C for the ts mutants was presumably not due to a greater rate of inactivation at 39 degrees C. Unlike WT VSV, which synthesized viral proteins equally well at all three temperatures, tsG31 had a reduced synthesis of all the structural proteins at 37 and 39 degrees C, compared with that at 31 degrees C; the formation of the M protein was most temperature sensitive. In addition, fractionation of the infected cells indicated that the incorporation of the M and N proteins into the cellular membranes was also disrupted at the higher, nonpermissive temperatures. Several characteristics of protein synthesis during tsG31BP infection at 39 degrees C distinguished this virus from tsG31: (i) no mature viral proteins were detected at 39 degrees C; (ii) several host proteins were [ill], suggesting that the virus was incapable of completely depressing host macromolecular synthesis; and (iii) a great proportion of the incorporated radioactivity was found in unusually high-molecular-weight proteins. In addition, at 37 degrees C, the tsG31BP virus showed a decreased synthesis of viral proteins and reduced assembly of the viral structural proteins.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Virus-Specific Proteins Synthesized in Cells Infected with RNA+ Temperature-Sensitive Mutants of Sindbis Virus

All Sindbis virus temperature-sensitive mutants defective in "late" functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that (3)H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive.     

Maturation of dimeric viral RNA of Moloney murine leukemia virus.

We have analyzed the dimeric RNA present in Moloney murine leukemia virus (MoMuLV) particles. We found that the RNA in newly released virions is in a conformation different from that in mature virions, since it has a different electrophoretic mobility in nondenaturing agarose gels and dissociates into monomers at a lower temperature. On the basis of these results, we suggest that the RNA initially packaged into nascent virions is already dimeric but that the dimer undergoes a maturation process after the virus is released from the cell. In further experiments, we tested the possibility that this maturation event is linked to the maturation cleavage of the virion proteins, which is catalyzed by the viral protease (PR). We found that the dimeric RNA isolated from PR- mutant virions resembles that from immature virions: it has a lower electrophoretic mobility and a lower sedimentation rate, and it also dissociates at a lower temperature than does RNA from mature wild-type virions. When Kirsten sarcoma virus is rescued by a PR- mutant or by a somewhat leaky cysteine array mutant of MoMuLV, its RNA also exhibits a electrophoretic mobility lower than that in the wild-type pseudotype. These results suggest that the maturation of dimeric RNA in released virus particles requires the cleavage of the Gag precursor and the presence of an intact cysteine array in the released nucleocapsid protein.     

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.     

Polyoma Pseudovirions I. Sequence of Events in Primary Mouse Embryo Cells Leading to Pseudovirus Production

The relationship of the intracellular events leading to the production of polyoma pseudovirions in primary mouse embryo cells has been investigated. Replication of polyoma deoxyribonucleic acid (DNA) began 18 hr after infection. Assembly of viral capsid protein occurred 12 hr later. Intracellular fragments of host cell DNA, of the size found in pseudovirions, were first detected 36 hr after infection. The amount of intracellular 14S host DNA that was produced during infection was seven times greater than the amount of polyoma DNA synthesized. The relative pool sizes of polyoma DNA and 14S DNA at the time of virus assembly may dictate the amounts of polyoma virus and pseudovirus produced.