下调细胞P21WAF1/CIP1基因表达可抑制单纯疱疹病毒Ⅱ型复制

为了揭示细胞P21蛋白在单纯疱疹病毒Ⅱ型（herpes simplex virus type 2, HSV-2）复制中的作用,通过用HSV-2感染和感染前用特异性小干扰RNA (small interfering RNA,siRNA) 抑制P21基因表达,应用Western 印迹方法检测宿主细胞和病毒蛋白水平,用终点滴定法测定病毒半数组织培养感染量（50% tissue culture infectious dose, TCID50）,以及观察感染细胞的细胞病变效应（cytopathic effect, CPE）等3个方面,揭示细胞P21蛋白水平的变化对病毒复制的影响.结果表明,HSV-2在细胞内复制时可引起P21蛋白水平增高;而用特异性siRNA下调细胞P21基因表达时,可显著地抑制HSV-2 gB蛋白水平,减少培养细胞上清液中病毒TCID50.提示P21蛋白对HSV-2的复制具有重要的作用.     

SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis

Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5' and 3' untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5'-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5'-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5'-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Requirements for CEACAMs and cholesterol during murine coronavirus cell entry

Previous reports have documented that cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during infection by the enveloped coronavirus murine hepatitis virus (MHV). To move toward a mechanistic understanding of these phenomena, we used growth media enriched with methyl-beta-cyclodextrin or cholesterol to reduce or elevate cellular membrane sterols, respectively. Cholesterol depletions reduced plaque development 2- to 20-fold, depending on the infecting MHV strain, while supplementations increased susceptibility 2- to 10-fold. These various cholesterol levels had no effect on the binding of viral spike (S) proteins to cellular carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, rather they correlated directly with S-protein-mediated membrane fusion activities. We considered whether cholesterol was indirectly involved in membrane fusion by condensing CEACAMs into "lipid raft" membrane microdomains, thereby creating opportunities for simultaneous binding of multiple S proteins that subsequently cooperate in the receptor-triggered membrane fusion process. However, the vast majority of CEACAMs were solubilized by cold Triton X-100 (TX-100), indicating their absence from lipid rafts. Furthermore, engineered CEACAMs appended to glycosylphosphatidylinositol anchors partitioned with TX-100-resistant lipid rafts, but cells bearing these raft-associated CEACAMs were not hypersensitive to MHV infection. These findings argued against the importance of cholesterol-dependent CEACAM localizations into membrane microdomains for MHV entry, instead suggesting that cholesterol had a more direct role. Indeed, we found that cholesterol was required even for those rare S-mediated fusions taking place in the absence of CEACAMs. We conclude that cholesterol is an essential membrane fusion cofactor that can act with or without CEACAMs to promote MHV entry.     

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.     

TVB receptors for cytopathic and noncytopathic subgroups of avian leukosis viruses are functional death receptors

The identification of TVB(S3), a cellular receptor for the cytopathic subgroups B and D of avian leukosis virus (ALV-B and ALV-D), as a tumor necrosis factor receptor-related death receptor with a cytoplasmic death domain, provides a compelling argument that viral Env-receptor interactions are linked to cell death (4). However, other TVB proteins have been described that appear to have similar death domains but are cellular receptors for the noncytopathic subgroup E of ALV (ALV-E): TVB(T), a turkey subgroup E-specific ALV receptor, and TVB(S1), a chicken receptor for subgroups B, D, and E ALV. To begin to understand the role of TVB receptors in the cytopathic effects associated with infection by specific ALV subgroups, we asked whether binding of a soluble ALV-E surface envelope protein (SU) to its receptor can lead to cell death. Here we report that ALV-E SU-receptor interactions can induce apoptosis in quail or turkey cells. We also show directly that TVB(S1) and TVB(T) are functional death receptors that can trigger cell death by apoptosis via a mechanism involving their cytoplasmic death domains and activation of the caspase pathway. These data demonstrate that ALV-B and ALV-E use functional death receptors to enter cells, and it remains to be determined why only subgroups B and D viral infections lead specifically to cell death.     

Partial antiviral activities of the Asn631 chicken Mx against newcastle disease virus and vesicular stomatitis virus

Conflicting data existed for the antiviral potential of the chicken Mx protein and the importance of the Asn631 polymorphism in determination of the antiviral activity. In this study we modified the chicken Mx cDNA from the Ser631 to Asn631 genotype and transfected them into COS-I cells, chicken embryonic fibroblast (CEF) or NIH 3T3 cells. The Mx protein was mainly located at the cytoplasm. The transfected cell cultures were challenged with newcastle disease virus (NDV) or vesicular stomatitis virus (VSV), cytopathic affect (CPE) inhibition assay showed that the times for development of visible and full CPE were significantly postponed by the Asn631 cDNA transfection at 48 h transfection, but not by the Ser631 cDNA transfection. Viral titration assay showed that the virus titers were significantly reduced before 72 h postinfection. CEF cells was incubated by the cell lysates extracted from the COS-I cells transfected with pcDNA-Mx/Asn631, could resist and delayed NDV infection. These data suggested the importance of the Asn631 polymorphism of the chicken Mx in determination of the antiviral activities against NDV and VSV at early stage of viral infection, which were relatively weak and not sufficient to inhibit the viral replication at late stage of viral infection.     

Immunogenicity of cytopathic and noncytopathic viral vectors

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.     

Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.     

Inhibition of arbovirus assembly by cycloheximide

Addition of cycloheximide (100 μg/ml) to cultures of chick cells infected with Semliki Forest virus (SFV) halted subsequent increase in virus titers. When added after 4 hr of infection, the drug had no effect on the rate of viral ribonucleic acid (RNA) synthesis, although marked inhibition of protein synthesis was seen. All of the previously identified forms of SFV RNA were seen in the drug-treated cells at higher concentrations than were present in untreated controls. The latter observation appeared to result from a failure to form viral “cores” or nucleocapsids in the cycloheximide-treated cells, resulting in sequestration of viral RNA intracellularly. The failure to form new virus cores was correlated with the failure of type II cytopathic vacuoles to appear in thin sections. Virus budding from the cell surface and the formation of type I cytopathic vacuoles persisted in cycloheximide-treated cells. The cellular pool of the major protein present in the virus core appeared to be small. None of this protein was found in a free pool in cytoplasm. The results indicated that, in the presence of cycloheximide, virus assembly was impaired because of the small size of the cellular pool of the major protein required for virus core formation.     

Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10⁵ PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.     

Transdominant inhibition of bovine viral diarrhea virus entry

Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100- to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor.     

Soluble receptor-mediated targeting of mouse hepatitis coronavirus to the human epidermal growth factor receptor

The mouse hepatitis coronavirus (MHV) infects murine cells by binding of its spike (S) protein to murine CEACAM1a. The N-terminal part of this cellular receptor (soR) is sufficient for S binding and for subsequent induction of the conformational changes required for virus-cell membrane fusion. Here we analyzed whether these characteristics can be used to redirect MHV to human cancer cells. To this end, the soR domain was coupled to single-chain monoclonal antibody 425, which is directed against the human epidermal growth factor receptor (EGFR), resulting in a bispecific adapter protein (soR-425). The soR and soR-425 proteins, both produced with the vaccinia virus system, were able to neutralize MHV infection of murine LR7 cells. However, only soR-425 was able to target MHV to human EGFR-expressing cancer cells. Interestingly, the targeted infections induced syncytium formation. Furthermore, the soR-425-mediated infections were blocked by heptad repeat-mimicking peptides, indicating that virus entry requires the regular S protein fusion process. We conclude that the specific spike-binding property of the CEACAM1a N-terminal fragment can be exploited to direct the virus to selected cells by linking it to a moiety able to bind a receptor on those cells. This approach might be useful in the development of tumor-targeted coronaviruses.     

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.     

RNA干扰抑制登革病毒复制的研究

为了研究RNA干扰(RNAi)对Ⅰ型登革病毒(DENV-1)在白纹伊蚊C6/36细胞内复制的影响,本研究设计并合成针对I型登革病毒Pr M基因的小干扰RNA,以脂质体法转染入C6/36细胞后,用DENV-1感染已转染的细胞,观察细胞病变效应,MTT法检测细胞存活率,荧光定量RT-PCR检测登革病毒RNA含量。结果表明:转染siRNA的C6/36细胞在受登革病毒攻击7天后仍无明显细胞病变效应,细胞存活率比对照组提高2.26倍,细胞内登革病毒RNA拷贝数比对照组降低约97.54%。说明利用RNA干扰技术能有效抑制登革病毒核酸在C6/36细胞内复制,并对细胞具有一定保护作用,为登革热的防治提供了新的思路。     

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.     

Suppression of coronavirus replication by inhibition of the MEK signaling pathway

We previously demonstrated that infection of cultured cells with murine coronavirus mouse hepatitis virus (MHV) resulted in activation of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway (Y. Cai et al., Virology 355:152-163, 2006). Here we show that inhibition of the Raf/MEK/ERK signaling pathway by the MEK inhibitor UO126 significantly impaired MHV progeny production (a reduction of 95 to 99% in virus titer), which correlated with the phosphorylation status of ERK1/2. Moreover, knockdown of MEK1/2 and ERK1/2 by small interfering RNAs suppressed MHV replication. The inhibitory effect of UO126 on MHV production appeared to be a general phenomenon since the effect was consistently observed in all six different MHV strains and in three different cell types tested; it was likely exerted at the postentry steps of the virus life cycle because the virus titers were similarly inhibited from infected cells treated at 1 h prior to, during, or after infection. Furthermore, the treatment did not affect the virus entry, as revealed by the virus internalization assay. Metabolic labeling and reporter gene assays demonstrated that translation of cellular and viral mRNAs appeared unaffected by UO126 treatment. However, synthesis of viral genomic and subgenomic RNAs was severely suppressed by UO126 treatment, as demonstrated by a reduced incorporation of [3H]uridine and a decrease in chloramphenicol acetyltransferase (CAT) activity in a defective-interfering RNA-CAT reporter assay. These findings indicate that the Raf/MEK/ERK signaling pathway is involved in MHV RNA synthesis.     

Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.     

Mechanism of Mengo Virus-Induced Cell Injury in L Cells: Use of Inhibitors of Protein Synthesis to Dissociate Virus-Specific Events

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.     

Pseudotype virions formed between mouse hepatitis virus and lactate dehydrogenase-elevating virus (LDV) mediate LDV replication in cells resistant to infection by LDV virions.

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) and mouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA which productively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV. These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistently LDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodies to the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particles containing the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infection by MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of the LDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse. Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cells possess a surface component that acts as an LDV receptor.