Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation. / Medical Sciences

Rabies virus (RABV) subverts host neuronal circuitry to gain access to the brain where it causes generally incurable, lethal encephalitis. The single glycoprotein (G) dictates two defining steps for infection and neuroinvasion: receptor-mediated endocytosis and transport of virus. We generate two recombinant VSV (rVSV) clones that genetically incorporate G (rVSV RABV G) from the fixed RABV strains, SAD B19 and CVS, to study internalization into epithelial cells and compartmentalized primary cultures of peripheral neurons. Through the use of chemical inhibitors and markers for specific endocytic routes, we demonstrate that the predominant RABV entry route in both epithelial and neuronal cells is dynamin- and clathrin- dependent. Viral endocytosis is mediated by actin-dependent, partially coated clathrin pits as evidenced by live high resolution confocal microscopy of envelopment in epithelial cells and transmission electron micrographs in neuronal and non-neuronal cells. Thus, we corroborate the hypothesis that particle size is the sole viral determinant of actin-dependence of coated pits. Through a combination of high resolution microscopy and infectivity-based approaches, we link molecular mechanisms of viral uptake at the single particle level to productive infection. Targeted pharmacological disruption of endosomal acidification at the neurites or cell bodies of peripheral neurons demonstrates that fusion and viral genome release at the cell body, the site of replication, is a prerequisite for infection. This work extends the current understanding of RABV entry by providing a detailed characterization of endocytosis from the plasma membrane to the site of fusion and correlating it with establishment of infection into neuronal populations relevant for pathogenesis in vivo. / Biology, Molecular and Cellular

The gastrointestinal tract is home to trillions of bacteria that interact with each other and with the host’s mucosal immune system. Obligate and facultative anaerobes thrive in the small and large intestine. While many of these bacteria have beneficial relationships with their host, opportunistic pathogens can bloom in times of inflammation and prolong disease. Klebsiella pneumoniae is an opportunistic pathogen that is part of the gut microbiota in many healthy individuals. Here we explore the population dynamics of K. pneumoniae in the gastrointestinal tract in mouse models of health and disease. To assess the role of intra-species genomic diversity in interactions with the host, a mouse K. pneumoniae isolate and three human clinical isolates from human stool, sputum, and urine were studied in the T-bet-/- Rag2-/- and dextran sodium sulfate models of ulcerative colitis and in a mouse model of systemic neonatal infection. Regardless of host origin, isolate site source, or genomic differences, all four K. pneumoniae isolates were able to stimulate colonic inflammation. However, only exposure to the murine K. pneumoniae isolate, and not human clinical isolates, led to neonatal death. In addition, this murine isolate correlated with differential shifts in levels of other Enterobacteriaceae species in the colon. Murine K. pneumoniae was found in higher amounts in host mesenteric lymph nodes compared to human isolates, suggesting subtle strain-based differences that affect response to K. pneumoniae in the gastrointestinal tract. A defining feature of K. pneumoniae is its mucoid capsular polysaccharide coat. To begin investigating a role for K. pneumoniae’s capsule in the gastrointestinal tract, K. pneumoniae biogeography was assessed in gnotobiotic mice. Encapsulated K. pneumoniae were outcompeted by naturally arising variants with reduced capsule throughout the gastrointestinal tract of gnotobiotic mice, except in the distal small intestine. This portion of the small intestine was also the region with the highest host Paneth cell antimicrobial peptide expression. Micro-injection of K. pneumoniae into small intestinal organoids revealed increased growth of encapsulated K. pneumoniae in the presence of α-defensins and decreased survival of K. pneumoniae with reduced capsule production. Capsule also conferred an advantage when K. pneumoniae was part of a diverse microbiota. Competition assays between K. pneumoniae and Escherichia coli showed enhanced survival of encapsulated K. pneumoniae compared to isolates with reduced capsule production. These data suggest that host AMPs, in combination with bacterial-bacterial interactions, shape population dynamics of K. pneumoniae and select for encapsulated K. pneumoniae throughout the gastrointestinal tract. Overall, these studies provide a foundation for understanding interactions between K. pneumoniae, the host, and other bacteria in the gastrointestinal tract. / Medical Sciences

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation. / Medical Sciences

In vitro protein synthesis in Vibrio costicola (polyuridilic acid (poly(U)) -directed incorporation of phenylalanine) was studied. As shown previously (Kamekura and Kushner, 1984), Cl$\sp-$ ions inhibited protein synthesis. The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. Washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. The main site of action of Cl$\sp-$ in this system is on the binding of ribosomes to the messenger RNA. I studied the protein synthesizing machinery of V. costicola using cell-free protein synthesis systems directed by natural mRNAs. One system, the endogenous system, was directed by a mixture of unidentified messengers present in V. costicola, the mRNAs endogenous to these cells. In another system, I have shown that the viral mRNA, that of the coliphage R17, directs the incorporation of ($\sp $C) valine with cellular extracts of V. costicola. The ionic requirements of both systems were similar; both were most active at NH$\sb4\sp+$ (as ammonium glutamate) and Mg$\sp{++}$ concentrations of 250-300 mM and 8 mM, respectively. As was the case with the poly(U) system, Cl$\sp-$ also inhibited protein synthesis in these systems. Added sodium or potassium glutamate inhibited the R17 RNA system but stimulated the endogenous system. It is believed that Cl$\sp-$ is more deleterious to the initiation than to the elongation of protein synthesis, and that glutamate stimulates elongation. Betaine stimulated the activity of both systems, but did not show the same protective effects against Cl$\sp-$ as those observed in the poly(U) system. I have successfully isolated a crude preparation of initiation factors of V. costicola. These factors were released from the ribosomes at a NH$\sb4$Cl concentration much higher than that required for the isolation of the initiation factors of E. coli (1.0 M). Cl$\sp-$ was more inhibitory to the retention of ($\sp $C) fmet-tRNA on nitrocellulose filters, than to the retention of ($\sp3$H) R17 RNA; the retention of these substances was presumably due to the action of IF$\sb2$ and IF$\sb3$, respectively. The enzymes choline dehydrogenase and betaine aldehyde dehydrogenase, which are involved in the synthesis of betaine from choline, were identified in V. costicola. Both enzymes are probably inducibly formed in the presence of choline and seem to be osmoregulated. V. costicola accumulates betaine in response to increasing external NaCl; the intracellular betaine concentration in cells grown in media containing 1.0 and 3.0 M NaCl is 0.25 and 1.2 M, respectively. This accumulation of betaine is also affected by the external concentration of choline. At the lowest choline concentration (0.002%), there is no difference in betaine concentration between cells grown in media containing 0.5, 1.0 and 2.0 M NaCl, while cells grown in the presence of 3.0 M NaCl have only slightly more (0.4 M). These results suggest that betaine may not be the only compatible solute in V. costicola and/or that it may be synthesized through an alternative pathway, and that these cells may also employ other means to maintain turgor pressure. (Abstract shortened by UMI.)

During sporulation, Baccilus thuringiensis subsp. kurstaki produces a crystalline inclusion body which is toxic upon ingestion by susceptible Lepidopteran larvae. The major component of crystals from Lepidopteran-specific subspecies of B. thuringiensis is a 130-kDa protein, protoxin. Following ingestion by susceptible larvae, protoxin is proteolyzed to yield a 58-70 kDa toxic fragment, toxin. In the present study, a simplified procedure was used for isolating and purifying toxin generated by the tryptic digestion of protoxin from B. thuringiensis subsp. kurstaki HD-73. Characterization of this toxin showed that it is derived from the N-terminal half of the protoxin molecule. The toxin is insoluble at neutral pH values but is moderately soluble at alkaline values above pH 9. Application of several spectroscopic and theoretical procedures to the purified toxin showed that the protein is composed of approximately equal amounts of a $\alpha$-helix, $\beta$-sheet and random coil structures. The tertiary structure of toxin was shown to be comprised of two primary domains; these domains correspond to the toxic and specificity (or binding) domains predicted from analysis of protoxin gene nucleotide sequences. Evidence was obtained that at least one additional domain is present as a structural component of the C-terminal specificity domain. Both the toxic moiety within the protoxin molecule and free toxin were found to be unusually resistant to unfolding by chemical denaturants and to proteolysis. In contrast, the C-terminal half of protoxin could be readily unfolded and was extremely susceptible to proteolytic digestion. The unfolded protoxin and unfolded toxin were shown to refold rapidly into their native and biologically active conformations. Evidence was obtained that the conformation of the toxic moiety of protoxin is very similar to the conformation of toxin. Chemical modification of the cysteine and lysine residues in the protoxin did not affect the biological activity of the protein. However, the introduction of positive, negative or neutral groups onto these residues had a large effect on the solubility of the protein. These results, along with the results obtained from the unfolding/folding, studies, strongly indicate that the primary function of the C-terminal half of the protoxin molecule is to promote the formation of a stable crystal.

In this study, nucleotide and amino acid sequence analyses were conducted in the P3 region of the Enterovirus 70 genome. Nucleotide and amino acid sequence alignments show that Enterovirus 70 is a picornavirus and is closely related to polioviruses and coxsackieviruses. The 3D$\sp{\rm pol}$ and 3B(VPg) of Enterovirus 70 show higher similarity to the corresponding regions of Poliovirus 1, while two other regions, 3A and 3C$\sp{\rm pro}$, are more closely related to Coxsackievirus B3 sequences. These observations suggest that the P3 region of Enterovirus 70 diverged from poliovirus like sequences. For the two regions which show more similarity to Coxsackievirus B3 like sequences, there is some evidence that this could be the result of recombination, in the P3 region, between an the ancestral sequence to Enterovirus 70 and a coxsackievirus like genome. The importance of the P3 region in transcription, translation, and replication suggests that these recombinational events may be responsible for some of the biological properties of Enterovirus 70, particularly its temperature sensitive nature and broader host range. (Abstract shortened by UMI.)

The occurrence of multiple cellulase components of Trichoderma harzianum E58 and the implications of their existence on the hydrolysis of cellulose were examined. A single commercial enzyme preparation, Novo-Celluclast, showed different extents of hydrolysis of several cellulosic substrates over time. The filter paper activities of six batches of T. harzianum E58 showed poor correlation with the ability of these enzymes to hydrolyze other cellulosic substrates over extended periods of time. Hydrolysis of a single substrate by a single enzyme preparation resulted in similar slopes in reducing sugar production with enzyme concentration, between one-hour and twenty-four-hour hydrolyses. The multiplicity of the cellulase components of T. harzianum E58 was examined, and the number of endoglucanase components and their specificities towards $\beta$-1,4-linkages were studied. Several types of endoglucanases were produced by the fungus. The role of the exoglucanase was examined using sub-saturation concentrations of T. harzianum E58 cellulase. No significant increase in hydrolysis was observed when purified exoglucanase was added to the cellulase mixture. The high proportion of non-specific endoglucanases and the need for an efficient endoglucanase-to-exoglucanase ratio are discussed in terms of a modified model for cellulose hydrolysis. (Abstract shortened by UMI.)

No description available.

In this study, a simple protocol was developed to test the survival of a human rotavirus (HRV), rhinovirus type 14 (RV-14), and parainfluenzavirus type 3 (HPIV-3) as well as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on human hands. HPIV-3 lost nearly all of its infectivity within the first 60 minutes on the fingerpads. In contrast to this, nearly 16% of infectious RV-14 could be recovered even after 3 hours. The poor survival of HPIV-3 on human hands prompted us to investigate its survival on stainless steel disks (1 cm in diam.) under ambient conditions. The ability of the two respiratory viruses to survive well on non-porous inanimate surfaces indicates that, once contaminated, these surfaces may act as a potential virus source. To study the patterns of virus transfer, the following three experimental models were developed and tested: finger-to-finger, finger-to-disk and disk-to-finger. The results suggest that human hands and environmental surfaces, singly or in combination, have the potential to spread rotaviral infections, particularly in institutional settings. The comparatively rapid loss of HPIV-3 infectivity on hands may reduce their potential as vehicles for transmission. These results also suggest a role for environmental surfaces in the contamination of hands with respiratory viruses. We compared the efficiency of paper-, cloth- and an electric blow dryer in further reducing the level of infectious rotavirus and E. coli remaining on fingerpads washed with either 70% isopropanol, Savlon in water (1:200), an unmedicated liquid soap, or tap water alone. Irrespective of the hand-washing agent used, warm air drying produced the greatest and the cloth the smallest reduction in the numbers of both test organisms. These findings indicate the importance of the proper drying of washed hands, particularly when less effective hand-washing agents are used. The results of this study show that HRV, RV-14 and S. aureus can survive on human hands long enough to permit their spread through hands. In contrast to this, parainfluenzaviruses and E. coli appeared to be limited in their capacity to survive on hands. The differences found in the efficacy of commonly used hand-washing agents against HRV and the bacteria tested point to the importance of testing such products by proper in vivo protocols using representative viruses. (Abstract shortened by UMI.)