John J. Holland

Receptor affinities as major determinants of enterovirus tissue tropisms in humans.

Abstract Virulent poliovirus was bound by cell-free homogenates of brain, spinal cord, and intestine from tissues of human fetuses and adult rhesus monkeys, but not detectably by homogenates of tissues in which the virus does not normally propagate in vivo (with few exceptions). This binding of virus was reversed by conditions which are known to reverse the binding of enteroviruses by HeLa cell receptors, but which are known not to reverse antibody inactivation. Receptor activity was found to be present in the microsome fraction of susceptible tissue homogenates, but not in insusceptible tissue microsome fractions. The nonneurovirulent attenuated vaccine LSc strain of type 1 poliovirus was not bound by brain microsome fractions or homogenates, but was bound by intestinal tissue homogenates. With several Coxsackie and ECHO viruses also, correlation was found between virus tissue tropisms and ability of tissue homogenates to bind virus in vitro . As has been shown for cultivated human cells, the enterovirus receptor substances in human tissues appear to be protein. It is shown that the poliovirus susceptibility of nonneural human tissues in cell culture is due to the in vitro appearance of receptor material which was not present in detectable amounts in the cells in vivo . Human amnion cells left on the amniotic membrane, or freshly trypsinized from the membrane were insusceptible to poliovirus and contained no detectable amounts of receptor substance. When these cells were removed from the membrane by trypsinization and cultivated as monolayers on glass, they produced receptor and became completely susceptible to poliovirus within 1 week. However, in vitro cultivation of intact amniotic membranes, with the cells in normal contact with the collagen membrane, failed to render them susceptible to poliovirus and other enteroviruses. Under these conditions of tissue culture, amnion cells also failed to produce detectable amounts of receptor protein. It is concluded that enterovirus tropisms are due to a large extent to affinity or lack of affinity between virus protein surfaces and a specific component of the surfaces of of the various cells of the body. The wide range of enterovirus susceptibility of most human cells after in vitro cultivation appears to be a consequence of receptor synthesis in vitro -a function which is normally repressed in most cells in their differentiated state in the body.

High-quality controlled trials on preventing episodes of back problems: systematic literature review in working-age adults.

BACKGROUND CONTEXT Back problems (BPs), with their cost and disability, are a substantial burden for individuals, employers, and society. PURPOSE This systematic review of controlled trials evaluates the effectiveness of interventions to prevent BP episodes in working age adults. DATA SOURCES We searched MEDLINE/EMBASE through May 2007, and COCHRANE/Trials Registry through August 22, 2008 using search terms of back pain, back injuries or sciatica, linked to prevention, control, workplace interventions, or ergonomics and searched article bibliographies. STUDY SELECTION For systematic review inclusion, articles had to describe prospective controlled trials of interventions to prevent BPs in working-age adults, with intervention assignment either to individual participants or preexisting groups. Of 185 articles identified as potentially relevant, 20 trials (11%) met inclusion criteria. DATA EXTRACTION Researchers extracted relevant information from controlled trials and graded methodological quality. Because of heterogeneity of trials, meta-analysis was not performed. RESULTS Only exercise was found effective for preventing self-reported BPs in seven of eight trials (effect size 0.39 to >0.69). Other interventions were not found to reduce either incidence or severity of BP episodes compared with controls. Negative trials included five trials of education, four of lumbar supports, two of shoe inserts, and four of reduced lifting programs. CONCLUSIONS Twenty high-quality controlled trials found strong, consistent evidence to guide prevention of BP episodes in working-age adults. Trials found exercise interventions effective and other interventions not effective, including stress management, shoe inserts, back supports, ergonomic/back education, and reduced lifting programs. The varied successful exercise approaches suggest possible benefits beyond their intended physiologic goals. LEVEL OF EVIDENCE Systematic review Level I evidence.

Irreversible eclipse of poliovirus by HeLa cells.

Abstract The binding of poliovirus to receptors of either intact or disrupted human cells results in an apparent eclipse of infectivity immediately upon adsorption. However, this is not a true eclipse, for full virus infectivity is restored by treatment with a number of agents (by very concentrated salt solutions, or by hydrogen bond-disrupting agents such as 8 M urea, or by very low pH). Of those agents capable of reversing the binding between virus and cell, only concentrated salt solutions (e.g., 6 M LiCl) did not also destroy the virus-binding ability of receptor protein, a result supporting the concept that electrostatic forces may be the major binding forces in specific adsorption of poliovirus and other enteroviruses. No evidence for disulfide bridging between cell and virus in adsorption or penetration was obtained using sulfhydryl compounds. Trypsin digestion, which readily destroyed poliovirus receptor activity of HeLa cell microsomes nevertheless failed to release virus already bound to receptor, indicating that adsorbed virus is in close proximity to the trypsin-sensitive receptor protein. Poliovirus attached to living HeLa cells, when tested at intervals for restoration of infectivity with 6 M LiCl or 8 M urea, lost infectivity exponentially upon incubation at 37° but not at 0°. This appears to be a “true eclipse,” and it did not occur following adsorption of virus to receptors of disrupted cells. This eclipse of virus parallels virus “penetration” as measured by loss of antiserum sensitivity of adsorbed virus at intervals. At no time was most of the cell-bound virus found to have penetrated beyond the cell surface (beyond antiserum sensitivity) and to have retained infectivity recoverable with salt or urea treatment. This finding suggested that poliovirus is eclipsed either at the cell surface or very soon after having left the surface. Attempts to block virus eclipse with metabolic inhibitors were not successful. A poliovirus-resistant HeLa cell population failed to eclipse virus at the normal rate, and most uneclipsed virus remained at the cell surface. Even disrupted HeLa cells were able to eclipse poliovirus infectivity, but only when the cells were disrupted after virus attachment to receptors. Finally, it was found that cell membrane fractions of HeLa cell homogenates (membranes which sediment at low speeds) adsorbed and eclipsed poliovirus, whereas microsomal membranes adsorbed, but failed to eclipse poliovirus. It is concluded that irreversible eclipse of poliovirus may occur at the cell surface following reversible attachment to cell receptors, and that one of the consequences of this eclipse may be loss of antiserum sensitivity of the virus-cell complex.

Nucleic acid and protein synthesis during poliovirus infection of human cells

Poliovirus infection of HeLa cells apparently prevents polymerization of host cell RNA on its DNA template, but most pre-existing host cell ribosomal RNA molecules remain intact during poliovirus replication. DNA-directed RNA synthesis catalysed by aggregate enzyme RNA polymerase was greatly inhibited in vitro in preparations from infected cells. The priming ability of DNA from infected cells was equal to that of normal cells when Escherichia coli RNA polymerase was employed. Attempts to bring HeLa cell aggregate enzyme RNA polymerase into solution and free it of DNA were largely unsuccessful. However, a small fraction of the polymerase activity could be “solubilized” by sonication, and the activity of this “solubilized” fraction was approximately the same when either normal or infected cells were used as the source of enzyme. HeLa cell protein synthesis was inhibited by poliovirus infection more rapidly than by actinomycin D, so that it could not be merely an indirect result arising from virus-induced suppression of messenger RNA synthesis. The mechanism by which virus depressed protein synthesis in infected cells was either weakly operative or non-operative in cell-free systems derived from infected cells.

Inhibition of host cell macromolecular synthesis by high multiplicities of poliovirus under conditions preventing virus synthesis

At an input multiplicity of 1,000 to 10,000 plaque-forming units (approximately 100,000 to over 1,000,000 physical particles) per cell, purified type 1 poliovirus caused rapid and profound inhibition of protein synthesis in HeLa cells and a less rapid but definite inhibition of RNA synthesis in the presence of 0–0025 M -guanidine. This concentration of guanidine completely prevents replication of the poliovirus RNA genome. Under these conditions cytopathic effects were visible within 6 hours and most cells were dead or dying within 10 to 24 hours. Puromycin and fiuorophenylalanine, at levels which inhibit protein synthesis and virus-directed RNA synthesis, failed to suppress or delay these high multiplicity effects on cell synthesis. It appears that some component(s) of the mature virus particle is capable of inhibiting host cell synthesis when added in sufficient quantity.

Inhibition of DNA-primed RNA synthesis during poliovirus infection of human cells

Abstract It was shown previously that poliovirus infection of HeLa cells leads within several hours to a drastic change in the base ratios of newly-synthesized RNA, and it was suggested that the synthesis of normal cell RNA is inhibited (Holland, 1961) . Data in press indicate that most RNA synthesis during polio-virus infection is virus-directed (Holland, 1963a) and that host cell-controlled RNA synthesis is in fact suppressed up to 90% during poliovirus infection (Holland, 1963b) . The present report concerns the mechanism by which virus suppresses normal cell RNA synthesis. It will be demonstrated that DNA extracted from infected cells is as capable of priming RNA synthesis with E. coli RNA polymerase as normal cell DNA. Even the “native” deoxyribonucleo-proteins of gently-disrupted normal and infected HeLa cells are equal in priming ability for the E. coli RNA polymerase described by Chamberlin and Berg (1962) . However, “aggregate enzyme” (DNA-protein complex containing RNA polymerase [Weiss, 1960]) extracted from infected cells shows only a small fraction of the RNA polymerizing activity of similar preparations extracted from normal cells. Baltimore and Franklin (1962) have recently reported a similar depression of aggregate enzyme activity in Mengo virus infection of mouse cells.

Interference between enteroviruses and conditions effecting its reversal

Abstract Interference between several immunologic types of enteroviruses was studied and found to involve a 10-fold to 100-fold reduction in the rate of synthesis of challenge virus RNA. Interference between equal multiplicities of virus was not induced unless interfering virus was allowed to replicate for approximately 1 hour before challenge, indicating that the phenomenon involves an intracellular event. Interference induced by a guanidine-sensitive virus was shown to be reversible when resistant cells were challenged with a guanidine-resistant mutant in the presence of guanidine. In addition, interference occurred normally when a mutant of the interfering virus was used as the challenge. These results support a hypothesis that this type of interference among virulent enteroviruses is due to competition for substrate or for replicating sites, and is not due to a stable interferon-like protein. When interfering virus was allowed to replicate for 3–4 1 2 hours prior to challenge with a homologous or heterologous guanidine-resistant virus, the replication of the second virus was not accelerated. These results demonstrate that superinfection of “primed” cells (those in which molecules involved in early viral synthesis were already present) did not accelerate replication of the challenge virus.

Evidence for cytoplasmic replication of poliovirus ribonucleic acid

Abstract Poliovirus-infected HeLa cells were fractionated into nuclear and cytoplasmic components, and the amount of infectious RNA extractable from each was determined. When guanidine was employed to prevent replication of viral RNA, crude preparations of nuclei contaminated with plasma membranes contained significant amounts of infectious RNA. But nuclei purified by sedimentation from dense sucrose solutions contained very little infectious RNA, although cytoplasmic fractions exhibited large quantities of infectious RNA. Furthermore, crude nuclear preparations contained very little infectious RNA several hours after cells were infected with proflavine-containing poliovirus and then exposed to light to inactivate uneclipsed, intact poliovirus which remained on plasma membrane receptors. It appears that most infecting virus or its infectious RNA migrates to sites in the cytoplasm rather than the nucleus in preparation for replication. When guanidine was not employed to prevent virus replication, or when it was removed following a long period of virus penetration, newly synthesized infectious RNA was again found mainly in the cytoplasmic fraction. When HeLa cells were treated with actinomycin, then infected with poliovirus, nearly all newly synthesized RNA (Pa32-labeled) appeared in the cytoplasm, and had base ratios identical to virus RNA. Less than 1% of the total RNA synthesized under these conditions appeared in purified nuclei, and this small amount of nuclear RNA did not have base ratios similar to poliovirus RNA. Finally, the new RNA polymerase which appears after poliovirus infection was not present in significant amounts in purified nuclei but was found almost exclusively in the cytoplasmic fraction. Since neither infectious RNA, nor RNA having virus base ratios, nor virus RNA polymerase, accumulates in the nucleus of infected cells prior to or during poliovirus replication, both the poliovirus RNA genome and its protein capsid are probably produced in the cytoplasm, but nuclear synthesis and very rapid migration to the cytoplasm cannot be rigorously excluded.

Altered base ratios in HeLa cell RNA during poliovirus infection

Abstract Ackermann et , al. (1959) reported that resting (non-dividing) HeLa cells infected with poliovirus produced large increments of protein and ribonucleic acid (RNA) as compared to uninfected controls. However, the incremental RNA exhibited base ratios similar to normal HeLa cell RNA, indicating that the bulk of the new RNA was not virus RNA. Salzman et , al. (1959) found no significant increase in RNA of HeLa cells infected with poliovirus when the cells were actively multiplying, rather than resting. The present report shows that the RNA in actively-multiplying HeLa cells infected with poliovirus has greatly altered base ratios. This shift in base ratio was detected by measuring P 32 incorporated into 2′, 3′ nucleotides of RNA during the period of virus replication. The shift is in the direction of the base composition of RNA obtained from purified poliovirus (Schaffer et , al. , 1960) .