Induction of the Chlamydia muridarum stress/persistence response increases azithromycin treatment failure in a murine model of infection.

Viable but noninfectious (stressed/persistent) chlamydiae are more resistant to azithromycin (AZM) in culture than are organisms in the normal developmental cycle. Chlamydia muridarum-infected mice were exposed to amoxicillin to induce the organisms to enter the persistent/stressed state and subsequently treated with AZM. AZM treatment failure was observed in 22% of persistently infected mice, with an average of 321,667 inclusion-forming units (IFU) shed after AZM treatment. Productively infected mice had a 9% rate of AZM treatment failure and shed an average of 12,083 IFU. These data suggest that stressed chlamydiaeare more resistant to frontline antichlamydial drugs in vivo.

Chlamydia muridarum enters a viable but non-infectious state in amoxicillin-treated BALB/c mice.

In culture, exposure to penicillin and other stressors induce chlamydiae to enter a non-infectious but viable state termed persistence. Chlamydiae may reenter their normal developmental cycle after stressor removal. Though aberrant RB similar to those present in culture models of persistence have been observed within infected tissues, the existence of persistent chlamydiae has not been definitively demonstrated in vivo. As a result, the role of persistent organisms in pathogenesis is undefined. In order to establish an experimentally tractable model of in vivo persistence, Chlamydia muridarum vaginally-infected mice were gavaged with either water or amoxicillin (amox). Vaginal swabs were collected for chlamydial titration and RNA isolated for quantification of pre-16s rRNA. Uterine tissue was analyzed by transmission electron microscopy (TEM). Although amox-treatment reduced vaginal shedding by >99%, C. muridarum pre-16s rRNA accumulation was unchanged by treatment. These data indicate that the amox-exposed organisms were viable but not infectious. Furthermore, TEM analyses demonstrated that inclusions in amox-treated animals contained primarily large, aberrant RB, but those observed in untreated control animals were normal. Collectively, these data suggest that amoxicillin treatment induces C. muridarum to enter the persistent state in vivo. This model also represents the first experimentally tractable animal model of chlamydial persistence.

Chlamydia persistence -- a tool to dissect chlamydia--host interactions.

Under stress, chlamydiae can enter a non-infectious but viable state termed persistence. In the absence of a tractable genetic system, persistence induction provides an important experimental tool with which to study these fascinating organisms. This review will discuss examples of: i) persistence studies that have illuminated critical chlamydiae/host interactions; and ii) novel persistence models that will do so in the future.

Interaction of herpes simplex virus type 2 (HSV-2) glycoprotein D with the host cell surface is sufficient to induce Chlamydia trachomatis persistence.

When presented with certain unfavourable environmental conditions, Chlamydia trachomatis reticulate bodies (RBs) enter into a viable, yet non-cultivable state called persistence. Previously, we established an in vitro C. trachomatis and herpes simplex virus type 2 (HSV-2) co-infection model. These data indicate that (i) viral co-infection stimulates chlamydial persistence, (ii) productive HSV replication is not required for persistence induction, and (iii) HSV-induced persistence is not mediated by any currently characterized anti-chlamydial pathway or persistence inducer. In this study we demonstrated that chlamydial infectivity, though initially suppressed, recovered within 44 h of co-infection with UV-inactivated HSV-2, demonstrating that HSV-induced persistence is reversible. Co-incubation of chemically fixed, HSV-2-infected inducer cells with viable, C. trachomatis-infected responder cells both suppressed production of infectious chlamydial progeny and stimulated formation of swollen, aberrantly shaped RBs. In addition, pre-incubation of viral particles with viral glycoprotein D (gD)-specific neutralizing antibody prevented co-infection-induced persistence. Finally, exposure of C. trachomatis-infected cells to a soluble, recombinant HSV-2 gD : Fc fusion protein decreased production of infectious EBs to a degree similar to that observed in co-infected cultures. Thus, we conclude that interaction of HSV gD with the host cell surface is sufficient to trigger a novel host anti-chlamydial response that restricts chlamydial development.

Increased ganglionic responses to substance P in hypertensive rats due to upregulation of NK(1) receptors.

Intravenous injection of substance P (SP) increases renal nerve firing and heart rate in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) by stimulating sympathetic ganglia. Blood pressure is increased in SHRs but lowered in WKYs. This study assesses the role of neurokinin-1 (NK(1)) receptors in mediating the ganglion actions of SP. Rats for functional studies were anesthetized and then treated with chlorisondamine. Renal nerve, blood pressure, and heart rate responses to intravenous injection of the NK(1) receptor agonist GR-73632 were similar but less than those to equimolar doses of SP in SHRs. GR-73632 only slightly increased renal nerve firing and heart rate and lowered blood pressure in WKYs. The NK(1) receptor antagonist GR-82334 (200 nmol/kg iv) blocked the ganglionic actions of GR-73632 and the pressor response to SP in SHRs. It reduced the renal nerve and heart rate responses by 52 and 35%. This suggests that the pressor response to SP is mediated by ganglionic NK(1) receptors and that NK(1) receptors also have a prominent role in mediating the renal nerve and heart rate responses to SP. Quantitative autoradiography showed that NK(1) receptors are more abundant in the superior cervical ganglia of SHRs. RT-PCR showed increased abundance of NK(1) receptor mRNA in SHRs as well. These observations suggest that the greater ganglionic stimulation caused by SP in SHRs is due to upregulation of NK(1) receptors.

Genomic organization of the human rod photoreceptor cGMP-gated cation channel beta-subunit gene.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel beta-subunit is complex, comprising non-overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213-218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100kb of genomic DNA on chromosome 16. The beta-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5kb of the upstream region were sequenced. Putative cis-elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Definition of the RRE binding and activation domains of the caprine arthritis encephalitis virus Rev protein.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus and is more distantly related to the human AIDS virus, HIV-1. Previous studies indicated that the CAEV Rev protein (Rev-C) functions as a trans-activator of mRNA cytoplasmic transport and expression. The function of Rev-C is mediated through an RNA element (RRE-C) present between nucleotides (nt) 7906 and 8110 in the CAEV env gene. In this study, RNA/protein immunoprecipitation experiments were used to demonstrate that Rev-C binds directly to the 204-nt RRE-C in vitro. Competition assays illustrate that this interaction is specific for the positive sense RRE-C RNA. Glutaraldehyde crosslinking studies demonstrate that the wildtype Rev-C protein can also form multimeric complexes in vitro. Deletions or amino acid alterations within the basic domain of Rev-C reduce affinity for the RRE and disrupt assembly of Rev-C multimers in vitro, indicating that this domain is involved in RRE binding and Rev multimer formation. Mutations within the leucine-rich domain of Rev-C do not greatly effect RRE-C binding or self-assembly. However, previous results demonstrate that some leucine-rich domain mutants are unable to trans-activate. These data are consistent with the hypothesis that the leucine domain is the effector domain of Rev-C.

Characterization of the temporal accumulation of minute virus of mice replicative intermediates.

We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65% of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10% of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer of similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.

A Rev protein is expressed in caprine arthritis encephalitis virus (CAEV)-infected cells and is required for efficient viral replication.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus human immunodeficiency virus 1 (HIV-1). Like other lentiviruses, the genome of CAEV contains multiple small ORFs that encode viral regulatory proteins. Sequence analysis of the CAEV genome and cDNAs generated from mRNA in infected cells has suggested that one of these ORFs encodes a protein (Rev-C) that is analogous to Rev of visna virus and HIV. Antibodies generated to a carboxy-terminal peptide of the rev ORF immunoprecipitate an 18-kDa protein from cells transfected with the Rev cDNA clone. Immunoprecipitation and immunofluorescence analysis of CAEV-infected ovine primary cells show that the product of the rev ORF is expressed during infection and localizes to the nucleolus of infected cells. Also, sera from CAEV-infected goats specifically immunoprecipitates an in vitro-translated product from the full-length Rev cDNA clone as well as that from the unique second open reading frame of Rev-C which shows that the Rev-C protein is expressed during natural CAEV infection of animals. Insertion of either a mutation that creates two stop codons in the unique second open reading frame of Rev-C or a mutation in the basic domain of Rev-C into the CAEV infectious molecular clone renders the virus unable to replicate in primary goat synovial membrane cells. Analysis of the RNA and proteins produced from both Rev-deficient clones indicates that they are defective in the accumulation of structural gene mRNAs in the cytoplasm as well as in synthesis of structural proteins compared to the wild-type CAEV clone. These data indicate that CAEV encodes a Rev protein that is required for efficient viral replication in culture.

The Rev protein of visna virus is localized to the nucleus of infected cells.

Visna virus is a lentivirus of sheep that is distantly related to the human lentivirus HIV-1. Like other lentiviruses, the genome of visna virus contains multiple small open reading frames that encode viral regulatory proteins. The product of one of these regulatory genes is the visna virus Rev protein, Rev-V. In this report, immunoprecipitation of visna virus-infected cells using a specific anti-Rev-V antibody, generated to a synthetic, carboxyl-terminal peptide of Rev-V, brings down a 22.5-kDa protein identical in size to the protein expressed from a functional Rev-V cDNA clone. Examination of the phosphorylation state of Rev-V indicates that it, unlike the Rev proteins of HIV-1 and CAEV, is not efficiently phosphorylated in infected cells. Cell fractionation and immunofluorescence analysis indicate that, in contrast to a previous report, Rev-V is strongly localized to the nucleus and concentrated in nucleoli of visna virus-infected cells. In addition, Rev-V localizes similarly in several different primary cells, in particular macrophages, infected with visna virus. These data indicate that the Rev-V protein is produced during visna virus infection and is localized to the nucleolus of the infected cell.

Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron.

mRNAs R1 and R2 of the parvovirus minute virus of mice encode the two essential viral regulatory proteins NS1 and NS2. Both RNAs are spliced between map units 44 and 46 (nucleotides 2280 and 2399); R2 RNAs are additionally spliced upstream between map units 10 and 39 (nucleotides 514 and 1989), using a nonconsensus donor and poor 3' splice site. The relative accumulation of R1 and R2 is determined by alternative splicing: there is twice the steady-state accumulation of R2 relative to that of R1 throughout viral infection, though they are generated from the same promoter and have indistinguishable stabilities. Here we demonstrate that efficient excision of the large intron to generate R2 is dependent on at least the initial presence, in P4-generated pre-mRNAs, of sequences within the downstream small intron. This effect is orientation dependent and related to the size of the intervening exon. Prior splicing of the small intron is unnecessary. Excision of the large intron is enhanced by changing its donor site to consensus, but only in the presence of the small intron sequences. Excision of the large intron is also enhanced by improving the polypyrimidine tract within its 3' splice site; however, in contrast, this change renders excision of the large intron independent of the downstream small intron. We suggest that sequences within the small intron play a primary role in efficient excision of the upstream large intron, perhaps as the initial entry site(s) for an element(s) of the splicesome, which stabilizes the binding of required factors to the polypyrimidine tract within the 3' splice site of the large intron.

Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product.

mRNAs R1 and R2 of the autonomous parvovirus minute virus of mice (MVM), which encode the viral nonstructural proteins NS1 and NS2, respectively, are processed in an ordered splicing pathway in which R2 is generated from mature spliced R1. Introduction of translation termination signals into these genes alters the processing of these RNAs; there is a significant (up to fourfold) increase in the accumulated steady-state levels of R1 relative to R2, when compared with wild-type levels, although the total accumulated levels of R1 plus R2 remain the same. The increase in accumulated R1 relative to R2 in mutant infected or transfected murine cells is independent of RNA stability and transport and decreases, in a polar manner, with the distance of the inserted termination signal from the shared initiation codon for NS1 and NS2 at nucleotide 260. The increased ratio of R1 to R2 is a consequence of the artificially introduced translation termination signals acting in cis rather than in the absence of a functional viral gene product. These mutations have an effect when they interrupt previously open reading frames in either exon of the spliced product R2. Nonsense mutations that are located in the second exon of R2 inhibit splicing of R1 to R2 only when they interrupt an open reading frame (ORF) that has the potential, after normal splicing, to be joined in-frame with the initiating AUG. These results suggest that nonsense mutations inhibit splicing of R1 to R2 by influencing the mechanism by which exons are defined in murine cells.

Accumulation of MVM gene products is differentially regulated by transcription initiation, RNA processing and protein stability.

The accumulation and stability of minute virus of mice (MVMp) RNA and protein as well as comparative strengths of the two viral promoters have been analyzed in highly synchronous infections of murine A9 fibroblasts. Results indicate that there is a temporal phasing of the accumulation of MVM RNA and protein: the RNA products of the P4 promoter appear prior to the products of the P38 promoter and NS1 and NS2 appear prior to the capsid proteins. Total and cytoplasmic spliced RNA accumulate similarly, although there is a lag in cytoplasmic accumulation of about 2 hr. Total RNA contains abundant unspliced R1 and R3 which are confined to the nucleus. There is no detectable difference in the ratio of the various spliced versions of each RNA species throughout infection. R2 accumulates faster and in a greater amount than R1 in both total and cytoplasmic RNA even though they are generated from the same promoter. During this same period, however, NS1 and NS2 accumulate to similar levels during 1-hr pulses. The stabilities of all MVM RNA species produced at both 9 and 12 hr postrelease are equivalent. Late in infection R3 accumulates faster and in greater amounts than the combined products of the P4 promoter, by approximately two- to threefold. This increase can be accounted for by an increase in the frequency of initiation from the P38 promoter, relative to P4, as assayed by nuclear run-on experiments. Therefore, the steady-state levels of the individual viral proteins during infection is controlled by specific regulation at the level of the initiation of transcription, RNA processing, and protein stability.