Giardiavirus-resistant Giardia lamblia lacks a virus receptor on the cell membrane surface.

Giardia lamblia virus (GLV) is a small nonenveloped double-stranded RNA virus that infects specifically the parasitic protozoan G. lamblia. Among the many collected strains of G. lamblia, a few turn out to be highly resistant to the virus infection. Two of these strains, Ac and JH, were subjected to electroporation with the RNA from GLV-infected G. lamblia WB strain. Subsequent studies indicated the presence of GLV double-stranded RNA and GLV protein in the electroporated and propagated cells. Virus particles, released by the transfected cells into the culture medium, were capable of infecting the virus-sensitive G. lamblia WB strain. When the WB cells were incubated with GLV at 4 degrees C and treated with the bifunctional cross-linking reagent disuccinimidyl suberate, little GLV protein was detectable inside the cells by immunofluorescent staining. However, patches of fluorescent granules were found on the membrane surface of the cells, suggesting cross-linking of the viruses with a certain membrane component(s). Similar treatment of the resistant strains Ac and JH showed no fluorescence either inside or outside of the cells. Two other closely related parasitic protozoa, Tritrichomonas foetus and Trichomonas vaginalis, cannot be infected by GLV via either viral infection or RNA transfection. The [35S]cysteine-labeled protein profiles in Triton X-114 extracts of G. lamblia WB, Ac, and JH were compared. The profile of the WB strain differs clearly from that of Ac and JH. It remains to be seen, however, whether this difference is related at all to the different susceptibilities to GLV infection.     

Transcription and Transport of Virus-Specific Ribonucleic Acids in African Green Monkey Kidney Cells Abortively Infected with Type 2 Adenovirus

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.     

Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication.

Stringent control of human immunodeficiency virus (HIV) replication was observed in the human monoblastoid cell line U937. A low-multiplicity infection of these cells by the LAV1 strain of HIV was productive for 2.5 days; then virus replication became restricted and no further evidence of virion production was observed. The dramatic decrease in HIV production was due in part of reduced accumulation of cytoplasmic viral RNA and occurred in the absence of evident cytopathic effects. In contrast, infected cells induced to differentiate by phorbol ester, vitamin D3, or lymphokine supernatant did not release markers of HIV despite the accumulation of significant levels of cytoplasmic viral RNA. HIV infection altered the pattern of c-myc RNA accumulation in U937 cells. Expression of this gene changes normally in response to the state of cellular differentiation; in infected cells the level of c-myc expression was correlated to the levels of viral RNA accumulation and not to cellular differentiation. These results suggest that restricted replication of HIV in monocytes might be an important mechanism of virus persistence and demonstrate a relationship between HIV replication and monocyte differentiation.     

Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.     

The physical state of herpes simplex virus DNA in infected human cells.

Treatment of herpes simplex virus type 1 (HSV-1)-infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm3), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm3) as well as in the intermediate fractions as determined by hybridization with 3H HSV complementary RNA. The viral DNA sequences of lower deisntiy did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds.     

Viral and Host Deoxyribonucleic Acid Synthesis in Shope Fibroma Virus-infected Cells as Studied by Means of High-Resolution Autoradiography

Incorporation of (3)H-thymidine by BSC-1 cells infected with Shope fibroma virus was studied by means of high-resolution electron microscopic radioautography. One-hour pulses with the radioactive precursor were given at various times after infection, during a one-step growth cycle of the virus. In the cytoplasm of infected cells, reacted grains occurred over foci of viroplasm; these foci are believed to represent the true sites of viral deoxyribonucleic acid (DNA) replication. Shope fibroma virus DNA synthesis began before 3 hr postinfection, reached a maximum at 8 to 9 hr, and then declined rapidly. It was demonstrated that the decline in (3)H-thymidine uptake is correlated with the onset of viral morphogenesis. In comparison with the noninfected culture, the nuclear labeling, which reflects host DNA metabolism, was slightly reduced by 4 hr postinfection. Inhibition became more marked as infection progressed, and host DNA synthesis was almost completely suppressed in late stages of viral development.     

Tissue tropism and target cells of bluetongue virus in the chicken embryo.

In situ cytohybridization was used to determine the tissue tropism and target cells for replication of bluetongue virus (BTV) in the developing chicken embryo. Hybridization with a biotinylated probe specific for segment 3 of BTV serotype 17 detected viral replication in embryos inoculated with U.S. serotypes 2, 10, 11, 13, and 17 or sheep blood containing a BTV field strain. At the final stages of infection, when the embryos were hemorrhagic, viral infection could consistently be detected in the brain, kidney, spinal cord, heart, lung, and liver, with the brain and kidney most severely affected. Other tissues, such as the retina, skin, tongue, and intestinal villi, also supported viral replication in some embryos. Greater concentrations of virus tended to be localized within epithelial cells, such as those lining the kidney tubules and tertiary bronchi of the lungs. Kinetics studies with BTV serotypes 11 and 17 and a field strain indicated that within 24 h after inoculation, viral replication occurred initially in the brain and kidney. By 48 h, viral replication was also detected in the lungs, heart, and spinal cord, with the liver being severely infected by 72 h. Low levels of hybridization could be detected in embryos infected with epizootic hemorrhagic disease virus, which is antigenically related to BTV.     

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.     

Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell

Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. I. Effect on synthesis of macromolecules by host cell. J. Bacteriol. 91:2317-2326. 1966.-The replication of mengovirus was studied in two strains of Novikoff (rat) hepatoma cells propagated in vitro. The replicative cycle in both strains required 6.5 to 7 hr. Infection resulted in a marked depression of ribonucleic acid (RNA) and protein synthesis by strain N1S1-63. Inhibition of RNA synthesis was reflected by a decrease in the deoxyribonucleic acid (DNA)-dependent RNA polymerase activity of isolated nuclei. Mengovirus had no effect on either protein or RNA synthesis or on the DNA-dependent RNA polymerase activity of a second strain, N1S1-67. The time course of viral-induced synthesis of RNA by cells was studied in cells treated with actinomycin D. It was first detectable between 2.5 and 3 hr after infection and continued until 6.5 to 7 hr. The formation of mature virus was estimated biochemically by measuring the amount of RNA synthesized as a result of viral infection which was resistant to degradation by ribonuclease in the presence of deoxycholate. Approximately 70% of the deoxycholate-ribonuclease-resistant RNA was located in mature virus, and the remainder was double-stranded. The formation of mature virus began about 45 min after viral-directed (actinomycin-resistant) synthesis of RNA was detectable in the cell, and only about 18 to 20% of the total RNA synthesized was incorporated into virus. Release of virus from cells began about 1 hr after maturation was first detectable. Release of virus from cells was accompanied by a loss of a large proportion of their cytoplasmic RNA and protein.     

Relationship Between Replication of Simian Virus 40 DNA and Specific Events of the Host Cell Cycle

The relationship between replication of simian virus 40 (SV40) DNA and the various periods of the host-cell cycle was investigated in synchronized CV(1) cells. Cells synchronized through a double excess thymidine procedure were infected with SV40 at the beginning or the middle of S, or in G(2). The first viral progeny DNA molecules were in all instances detected approximately 20 h after release from the thymidine block, independent of the time of infection. The length of the early, prereplicative phase of the virus growth cycle therefore depended upon the period of the cell cycle at which the cells were infected. Infection with SV40 was also performed on cells obtained in early G(1) through selective detachment of cells in metaphase. As long as the cells were in G(1) at the time of infection, the first viral progeny DNA molecules were detected during the S period immediately following, whereas if infection took place once the cells had entered S, no progeny DNA molecule could be detected until the S period of the next cell cycle. These results suggest that the infected cell has to pass through a critical stage situated in late G(1) or early S before SV40 DNA replication can eventually be initiated.     

Identification of a Plant Viral RNA Genome in the Nucleus

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.     

Mengovirus Replication in Novikoff Rat Hepatoma and Mouse L Cells: Effects on Synthesis of Host-Cell Macromolecules and Virus-specific Synthesis of Ribonucleic Acid

Novikoff cells (strain N1S1-67) and L-67 cells, a nutritional mutant of the common strain of mouse L cells which grows in the same medium as N1S1-67 cells, were infected with mengovirus under identical experimental conditions. The synthesis of host-cell ribonucleic acid (RNA) by either type of cell was not affected quantitatively or qualitatively until about 2 hr after infection, when viral RNA synthesis rapidly displaced the synthesis of cellular RNA. The rate of synthesis of protein by both types of cells continued at the same rate as in uninfected cells until about 3 hr after infection, and a disintegration of polyribosomes occurred only towards the end of the replicative cycle, between 5 and 6 hr. The time courses and extent of synthesis of single-stranded and double-stranded viral RNA and of the production of virus were very similar in both types of cells, in spite of the fact that the normal rate of RNA synthesis and the growth rate of uninfected N1S1-67 cells are about three times greater than those of L-67 cells. In both cells, the commencement of viral RNA synthesis coincided with the induction of viral RNA polymerase, as measured in cell-free extracts. Viral RNA polymerase activity disappeared from infected L-67 cells during the period of production of mature virus, but there was a secondary increase in activity in both types of cells coincidental with virus-induced disintegration of the host cells. Infected L-67 cells, however, disintegrated and released progeny virus much more slowly than N1S1-67 cells. The two strains of cells also differed in that replication of the same strain of mengovirus was markedly inhibited by treating N1S1-67 cells with actinomycin D prior to infection; the same treatment did not affect replication in L-67 cells.     

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.