Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Failure of reactivation of chick erythrocytes after fusion with temperature-sensitive mutants of mammalian cells arrested in G1

Two temperature-sensitive (ts) mutants of mammalian cell lines (AF8 and cs4D3) that arrest in G1 at the nonpermissive temperature were fused with chick erythrocytes and the induction of DNA synthesis was studied in the resulting heterokaryons. While both AF8 and cs4D3 could induce DNA synthesis in chick nuclei at the permissive temperature, they both failed to do so when arrested in G1 at the nonpermissive temperature. When S phase AF8 cells were fused with chick erythrocytes, chick nuclei were reactivated even if the heterokaryons were incubated at the temperature nonpermissive for AF8. A third ts mutant, ts111, that is blocked in cytokinesis but continues to synthesize DNA, reactivated chick nuclei at both permissive and nonpermissive temperature. It is concluded that chick erythrocyte reactivation depends on the presence of S phase-specific factors.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.     

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.

Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface.     

Homologous Interference Induced by Sindbis Virus

Homologous interference during Sindbis virus infection has been investigated. Prior infection of either chicken embryo fibroblast or BHK(21) cell cultures results in reduced yields of progeny virions of the superinfecting genotype. This reduction in yield results from a reduction in the number of cells in the cultures capable of producing the superinfecting genotype. The development of interference parallels the attachment kinetics of Sindbis virus. Interference requires an active viral genome since the activity is sensitive to inactivation by ultraviolet light, and an RNA(-) mutant, ts-24, fails to induce interference under nonpermissive conditions. However, ts-6, an RNA(-) mutant belonging to a different complementation group, and the RNA(+) mutants, ts-2 and ts-20, interfere at both permissive and nonpermissive temperatures.     

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.     

Morphology of BHK-21 Cells Infected with Sindbis Virus Temperature-Sensitive Mutants in Complementation Groups D and E

BHK-21 cells infected with temperature-sensitive mutants of Sindbis virus in complementation groups D and E differed in their appearance under nonpermissive conditions. Cells infected at nonpermissive temperature with virus defective in complementation group E had nucleocapsids attached in large numbers to the inside surface of the host plasma membrane. Infection with a group D mutant produced nucleocapsids that did not attach to the plasma membrane but rather remained free in the cell cytoplasm.     

Host range temperature-sensitive mutants of herpes simplex virus type 2.

Two small-plaque mutants of herpes simplex virus type 2 (HSV-2) (strain 333), whose growth at 39 C was blocked in certain cell types (cell-dependent temperature sensitivity), were compared compared with parental virus in a number of biological assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious, particles; under nonpermissive conditions, these mutant particles were able to interfere with the replication of wild-type HSV-2. The other mutant (no. 74), which is known to belong to a different complementation group, appeared to direct little virus DNA synthesis, even at the permissive temperature. Progeny production and virus DNA synthesis in cells infected by mutant 74 were delayed in comparison with wild-type virus-infected cells. Both mutants were found to be more sensitive to UV irradiation than the parental virus; this was especially marked in the case of mutant 74. Moreover, this mutant was found to have a high transforming efficiency at much lower doses of irradiation than those needed to abolish the cytopathic effect of wildtype HSV-2.     

BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus.

The process by which Sindbis virus excludes superinfecting homologous virus was investigated with the use of temperature-sensitive mutants. Mutants in two RNA-negative complementation groups were found to be defective in their ability to establish interference at the nonpermissive temperature. These mutants were unable to establish interference in a mixed infection (complementation), suggesting that both were defective in a common gene product. Homologous interference was found to block the replication of superinfecting virus after attachment, penetration, and translation of the nonstructural genes encoded in the virus RNA. The production of nonstructural gene products of superinfecting wild-type virus was found to enhance the replication of certain RNA- temperature-sensitive interfering viruses at the permissive and the nonpermissive temperature. The ability of certain RNA- mutants to establish homologous interference and to demonstrate enhanced growth after superinfection with wild-type virus was interpreted to produce a model implicating both virus and host components in the establishment of homologous interference and in the replication of Sindbis virus RNA.     

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.     

Simian virus 40 mutants carrying two temperature-sensitive mutations.

Five temperature-sensitive mutants of simian virus 40 containing two temperature-sensitive mutations were isolated. The double mutant of the A and D complementation groups, like the D mutants, failed to complement by conventional complementation analysis and did not induce host DNA synthesis at 40 degrees C. However, under conditions that suppressed the D defect, the A:D double mutant expressed only the A defect. Thus, viral DNA replication dropped rapidly after this mutant was shifted from permissive to restrictive temperatures. The A:D double mutant failed to transfrom at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. Double mutants of A:B, A:C, and A:BC complementation groups, like their A parent, were defective in viral DNA replication, in the induction of host DNA synthesis and in the transformation of secondary Chinese hamster lung cells at the nonpermissive temperature.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Approach to the Involvement of Influenza B Neuraminidase in the Cleavage of HA by Host Cell Protease Using Low pH-Induced Cell Fusion Reaction

ts7, a temperature-sensitive mutant defective in neuraminidase (NA) of influenza B/Kanagawa/73, lacks NA enzymatic activity at the nonpermissive temperature (37.5 C). When MDCK cells were infected with the mutant at the permissive temperature (32 C) and exposed to pH 5.2 medium, extensive cell fusion occurred. In contrast, at the nonpermissive temperature cells did not show cell fusion at all unless they were pretreated with trypsin, suggesting that at 37.5 C the hemagglutinin (HA) of ts7 is expressed at the cell surface in an uncleaved form. It was also found that the replacement of RNA segment 6 of ts7 with that of wild-type B/Lee resulted in the emergence of low pH-induced fusion activity as well as NA enzymatic activity at the incubation temperature of 37.5 C and that the addition of bacterial NA to the cultures infected with ts7 at 37.5 C early in infection brought about low pH-induced cell fusion. We suggest that the removal of neuraminic acid from the carbohydrate moiety of HA by NA is essential for the cleavage of HA by cellular protease.