Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear doublestranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE, was purified by construction of a fusion protein, GST-ParE, followed by glutathione- In order for a plasmid to be stably maintained in a bacterial population, each daughter cell must inherit at least one copy of the plasmid upon cell division. For high-copy-number plasmids, random segregation alone may be adequate for achieving stability. Low-copy-number plasmids, however, employ one or more genetic loci which encode mechanisms which stabilize the plasmid within a growing bacterial population (49). Several different plasmid stabilization mechanisms have been identified (19,31,56). One such system involves the selective killing of plasmid-free daughter cells of Escherichia coli. The hok/sok, srnB, and pnd loci of plasmids R1, F, and R483, respectively, encode cytotoxic proteins (Hok, SrnBЈ, and Pnd, respectively), which cause a ''ghost'' cell phenotype (14). Expression of the toxin is posttranscriptionally regulated by an antisense RNA, which blocks translation and exhibits a relatively short half-life (13, 15). In a plasmid-free segregant, the unstable antisense RNA is degraded, allowing for translation of the toxins and the subsequent death of the plasmid-free cell (52).In the case of another family of killing systems (23), which includes the ccd locus of F (2,9,20,22,47,51), the parD/pem locus of R1-R100 (4, 5, 39, 54), and the phd-doc system of prophage P1 (28), two proteins are involved, a toxin (CcdB, Kis/PemK, and Doc, respectively) and an antitoxin (CcdA, Kid/PemI, and Phd, respectively), which presumably is more susceptible to degradation in a plasmid-free cell (5, 20, 28). CcdA and PemI protein degradation has been shown to be mediated by the Lon protease (53, 55), while Phd degradation is mediated by the ClpXP serine protease (29). Thus, in the absence of the plasmid, sufficiently high levels of antitoxin are not maintained, and the toxin is able to kill the host cell. Cell death is accompanied by filamentation and has been shown for the ccd system to be caused by the ability of the CcdB protein to induce the ATP-dependent cleavage of DNA by gyrase (3). In the case of the F ccd and R1 parD systems, the toxin and antitoxin are transcribed from the same operon, which is autoregulated by the coordinated action of both proteins (9, 39, 51).RK2 is a broad-host-range plasmid (32) that despite a relatively low copy number of four to seven copies per chromosome (12) is stably maintained in a wide-range of gram-negative bacteria. Stabilization of RK2 is accomplished by at least two genetic regions of the plasmid utilizing different stabilization mechanisms. The psa (postsegregational arrest) locus, which has not been mapped, causes the growth inhibition of daughter cells which fail to inherit the plasmid (24). Factors encoded by this...

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno[coreceptor] and PSSMx4r5. Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log10 viral load change at week 8 was −2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were −1.2 for TF-ES or the Reflex Test and −1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p<0.0001). At week 8, the positive predictive value was 66% for TF-ES and 65% for both the Reflex test and UDS. Negative predictive values were 59% for TF-ES, 58% for the Reflex Test and 61% for UDS. In conclusion, genotypic tropism testing using UDS alone or a reflex strategy separated maraviroc responders and non-responders as well as a sensitive phenotypic test, and both assays showed improved performance compared to TPS alone. Genotypic tropism tests may provide an alternative to phenotypic testing with similar discriminating ability.

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